Supporting Information - PNAS · 2010. 8. 24. · Supporting Information Chakrabortee et al....
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Supporting Information Chakrabortee et al. 10.1073/pnas.1013560107 Supporting Information corrected 09/21/2010 SI Text Bioinformatics. The Uversky plot was obtained by analysis of the anhydrin amino acid sequence (AY340998) at www.pondr.com where the plot represents mean scaled hydropathy, hHi, against mean net charge, hRi, at neutral pH (1). The boundary line shown in Fig. 1A between unfolded and folded space is empirically de- fined by the equation hHi b ¼ ðhRiþ 1.151Þ∕2.785. Three inde- pendent predictors of protein disorder, VL-XT (www.pondr.com), IUPred (http://iupred.enzim.hu) and FoldIn- dex (http://bip.weizmann.ac.il/fldbin/findex), were used. Four al- gorithms indicate that anhydrin binds to DNA (http:// turing.cs.iastate.edu/PredDNA; http://bioinfo.ggc.org/bindn; http://lcg.rit.albany.edu/dp-bind; www.netasa.org/dbs-pred). Grand average hydropathy was calculated at http:// microbio1.biologie.uni-greifswald.de/cgi-bin/protalizer-web.cgi . Circular Dichroism and Fourier Transform Infrared Spectroscopy. For CD, protein concentration was 0.5 mg·ml −1 in water in a 0.1 cm pathlength cuvette. pcDNA3.1 was added at a mass ratio of 25∶1 (protein:DNA) in the presence of 10 mM MgCl 2 or 10 mM CaCl 2 and incubated for 30 min at 25 °C. Spectra were acquired at room temperature with a scan speed of 10 nm min −1 , a 0.5 nm wave- length interval and a 2 s integration time. Data was smoothed using the Savitzky–Golay algorithm and normalized to the same minimum value. For FTIR, lyophilized anhydrin was dissolved in deuterated water at 18.5 mg·ml −1 . To investigate DNA binding, anhydrin in deuterated water was mixed with pcDNA3.1 (25∶1) in nondeuterated water and 10 mM MgCl 2 or CaCl 2 . Each spectrum results from accumulation and averaging of 256 interferograms. Spectra of deuterated water or appropriate D 2 O-H 2 O mixtures in the case of the anhydrin-pcDNA3.1 sample were subtracted. A baseline correction was performed in amide I′ region (1;600–1;700 cm −1 ) assuming a linear baseline. Second-derivative spectra were calculated using Bruker OPUS 2.2 software. For presentation, the absorbance and the second-derivative spectra for MgCl 2 and pcDNA3.1, CaCl 2 , and CaCl 2 and pcDNA3.1 were multiplied by a factor of 3.5, 10 and 5, respectively. Vectors, Constructs, and Antibodies. The anhydrin cDNA sequence (2) was cloned into the pmCherry plasmid (Clontech; at the HindIII and KpnI sites) to generate anhydrin-mCherry, and with N-terminal His 6 tag and C-terminal FLAG sequences into both pET28a+ (Novagen; at the NdeI and SacI restriction sites) and pFLAG-CMV5a (Sigma; at the HindIII and KpnI restriction sites). All clones were validated by sequencing. pmCherry, pHM6, pHM6-PABPN1-A17, eGFPC1, eGFPC1-PABPN1-A17 were gifts of D.C. Rubinsztein and colleagues (CIMR, Cam- bridge, UK). Primary antibodies used for immunoblotting are monoclonal anti-Flag (1∶2;000, Sigma), antihistone H3 (1∶1;000, Cell Signaling Technology), anti-HA (1∶1;000 HA.11 clone 16B12 monoclonal, Covance), antitubulin (1∶3;000, Sigma), anti-GFP (1∶1;000, Invitrogen), and anti-His 6 (1∶200, Novagen). Secondary antibodies used are horseradish peroxidase-linked ECL antirabbit or antimouse IgG (1∶3;333, GE Healthcare). Recombinant Anhydrin. Growth of BL21 Rosetta (DE3) pLysS cells harboring anhydrin/pET28a+, expression and recovery of recom- binant protein using a nickel chelation column was as described (3). Protein was then subjected to two rounds of further purifi- cation using an AKTA FPLC system (GE Healthcare) by ion exchange chromatography (Mono S PC 1.6/5 column; linear gra- dient, 0–2 M NaCl) and size exclusion chromatography (SEC; Superdex 75 column). SEC was also used to determine the size of the protein with molecular weight markers as detailed in the legend to Fig. S1. Protein sequence was confirmed by MALDI- TOF mass spectrometry of GluC proteinase or tryptic digests on a Micromass MALDImicroMX and by PSDMX fragmentation. Mammalian Cell Culture and Transfection. T-REx293 (Invitrogen) cells were grown as described (4). Transient transfections were performed for 3 h with constructs as indicated in experiments using GeneJammer (Stratagene) and analyzed at 48 h posttrans- fection. 10 μM lactacystin was used to inhibit the proteasome (5). Western Blotting. Cell pellets were lysed on ice for 30 min in Laemmli buffer [62.5 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulphate, 5% β-mercaptoethanol, 10% glycerol, 0.01% bro- mophenol blue, Complete protease inhibitor cocktail (Roche Diagnostics)], boiled for 5 to 7 min and subjected to immunoblot- ting analysis, as previously described (6). Immunoprecipitation. Various plasmid constructs as indicated were cotransfected into mammalian cells (1 μg each). Cells were lysed in IP buffer (20 mM Tris-HCl pH7.2, 2 mM MgCl 2 , 150 mM NaCl, 0.5% NP40, Complete protease inhibitor cocktail) for 30 min on ice and centrifuged at 2,000 g for 5 min. For probing total cell lysates (inputs), an aliquot was taken from the supernatant. The remainder (500 μg to 1 mg protein) was incubated overnight at 4 °C with either anti-HA antibody (1∶100) or anti-Flag M2 af- finity gel (5 μg/ml; Sigma). Anti-HA immunocomplex was further bound to Dynabeads Protein G (Invitrogen) for 2 h at 4 °C. Fol- lowing three washes in IP buffer, immunoprecipitated proteins were either eluted with 0.1 M glycine pH2.5 and neutralized in 1.5 M Tris-HCl pH8.8 from protein G beads, or for anti-Flag pull- down, eluted directly by boiling in Laemmli buffer. Samples were subjected to immunoblot analysis, together with inputs. Subcellular Fractionation. Cytoplasmic and nuclear extracts were prepared from cells transfected with 1 μg pFLAG-CMV5a or pFLAG-CMV5a-anhydrin using a standard kit (Active Motif) as per manufacturer’s instructions. FRET Measurements. Image processing software was developed in IDL 6.0 (ITT Visual Information Solutions) to correct the FRET signal for cross-talk (donor emission into the acceptor channel) and cross-excitation (direct excitation of acceptor molecules at the donor excitation wavelength) (software available at http:// laser.ceb.cam.ac.uk). We used a FRET protocol that is capable of distinguishing the fraction of interacting versus noninteracting molecules which eliminates artefacts arising from changing inter- action stoichiometries, a method described in detail in a previous publication (7). Two quantities are evaluated: aFRET and dFRET , which signify the FRET efficiencies, normalized by the fraction of interacting acceptors and donors, respectively, from which infor- mation on the interaction stoichiometries can be inferred. For example, for dFRET the actual quantity calculated in each image pixel was Chakrabortee et al. www.pnas.org/cgi/doi/10.1073/pnas.1013560107 1 of 6
Transcript of Supporting Information - PNAS · 2010. 8. 24. · Supporting Information Chakrabortee et al....
Supporting InformationChakrabortee et al. 10.1073/pnas.1013560107
Supporting Information corrected 09/21/2010SI TextBioinformatics. The Uversky plot was obtained by analysis of theanhydrin amino acid sequence (AY340998) at www.pondr.comwhere the plot represents mean scaled hydropathy, hHi, againstmean net charge, hRi, at neutral pH (1). The boundary line shownin Fig. 1A between unfolded and folded space is empirically de-fined by the equation hHib ¼ ðhRi þ 1.151Þ∕2.785. Three inde-pendent predictors of protein disorder, VL-XT(www.pondr.com), IUPred (http://iupred.enzim.hu) and FoldIn-dex (http://bip.weizmann.ac.il/fldbin/findex), were used. Four al-gorithms indicate that anhydrin binds to DNA (http://turing.cs.iastate.edu/PredDNA; http://bioinfo.ggc.org/bindn;http://lcg.rit.albany.edu/dp-bind; www.netasa.org/dbs-pred).Grand average hydropathy was calculated at http://microbio1.biologie.uni-greifswald.de/cgi-bin/protalizer-web.cgi.
Circular Dichroism and Fourier Transform Infrared Spectroscopy. ForCD, protein concentration was 0.5 mg·ml−1 in water in a 0.1 cmpathlength cuvette. pcDNA3.1 was added at a mass ratio of 25∶1(protein:DNA) in the presence of 10 mMMgCl2 or 10 mMCaCl2and incubated for 30 min at 25 °C. Spectra were acquired at roomtemperature with a scan speed of 10 nm min−1, a 0.5 nm wave-length interval and a 2 s integration time. Data was smoothedusing the Savitzky–Golay algorithm and normalized to the sameminimum value. For FTIR, lyophilized anhydrin was dissolved indeuterated water at 18.5 mg·ml−1. To investigate DNA binding,anhydrin in deuterated water was mixed with pcDNA3.1 (25∶1) innondeuterated water and 10 mMMgCl2 or CaCl2. Each spectrumresults from accumulation and averaging of 256 interferograms.Spectra of deuterated water or appropriate D2O-H2O mixturesin the case of the anhydrin-pcDNA3.1 sample were subtracted.A baseline correction was performed in amide I′ region(1;600–1;700 cm−1) assuming a linear baseline. Second-derivativespectra were calculated using Bruker OPUS 2.2 software. Forpresentation, the absorbance and the second-derivative spectrafor MgCl2 and pcDNA3.1, CaCl2, and CaCl2 and pcDNA3.1 weremultiplied by a factor of 3.5, 10 and 5, respectively.
Vectors, Constructs, and Antibodies. The anhydrin cDNA sequence(2) was cloned into the pmCherry plasmid (Clontech; at theHindIII and KpnI sites) to generate anhydrin-mCherry, and withN-terminal His6 tag and C-terminal FLAG sequences into bothpET28a+ (Novagen; at the NdeI and SacI restriction sites) andpFLAG-CMV5a (Sigma; at the HindIII and KpnI restrictionsites). All clones were validated by sequencing. pmCherry,pHM6, pHM6-PABPN1-A17, eGFPC1, eGFPC1-PABPN1-A17were gifts of D.C. Rubinsztein and colleagues (CIMR, Cam-bridge, UK). Primary antibodies used for immunoblotting aremonoclonal anti-Flag (1∶2;000, Sigma), antihistone H3 (1∶1;000,Cell Signaling Technology), anti-HA (1∶1;000 HA.11 clone16B12 monoclonal, Covance), antitubulin (1∶3;000, Sigma),anti-GFP (1∶1;000, Invitrogen), and anti-His6 (1∶200, Novagen).Secondary antibodies used are horseradish peroxidase-linkedECL antirabbit or antimouse IgG (1∶3;333, GE Healthcare).
Recombinant Anhydrin.Growth of BL21 Rosetta (DE3) pLysS cellsharboring anhydrin/pET28a+, expression and recovery of recom-binant protein using a nickel chelation column was as described(3). Protein was then subjected to two rounds of further purifi-
cation using an AKTA FPLC system (GE Healthcare) by ionexchange chromatography (Mono S PC 1.6/5 column; linear gra-dient, 0–2 M NaCl) and size exclusion chromatography (SEC;Superdex 75 column). SEC was also used to determine the sizeof the protein with molecular weight markers as detailed in thelegend to Fig. S1. Protein sequence was confirmed by MALDI-TOFmass spectrometry of GluC proteinase or tryptic digests on aMicromass MALDImicroMX and by PSDMX fragmentation.
Mammalian Cell Culture and Transfection. T-REx293 (Invitrogen)cells were grown as described (4). Transient transfections wereperformed for 3 h with constructs as indicated in experimentsusing GeneJammer (Stratagene) and analyzed at 48 h posttrans-fection. 10 μM lactacystin was used to inhibit the proteasome (5).
Western Blotting. Cell pellets were lysed on ice for 30 min inLaemmli buffer [62.5 mM Tris-HCl pH 6.8, 2% sodium dodecylsulphate, 5% β-mercaptoethanol, 10% glycerol, 0.01% bro-mophenol blue, Complete protease inhibitor cocktail (RocheDiagnostics)], boiled for 5 to 7 min and subjected to immunoblot-ting analysis, as previously described (6).
Immunoprecipitation.Various plasmid constructs as indicated werecotransfected into mammalian cells (1 μg each). Cells were lysedin IP buffer (20 mM Tris-HCl pH7.2, 2 mM MgCl2, 150 mMNaCl, 0.5% NP40, Complete protease inhibitor cocktail) for 30min on ice and centrifuged at 2,000 g for 5 min. For probing totalcell lysates (inputs), an aliquot was taken from the supernatant.The remainder (500 μg to 1 mg protein) was incubated overnightat 4 °C with either anti-HA antibody (1∶100) or anti-Flag M2 af-finity gel (5 μg/ml; Sigma). Anti-HA immunocomplex was furtherbound to Dynabeads Protein G (Invitrogen) for 2 h at 4 °C. Fol-lowing three washes in IP buffer, immunoprecipitated proteinswere either eluted with 0.1 M glycine pH2.5 and neutralized in1.5 M Tris-HCl pH8.8 from protein G beads, or for anti-Flag pull-down, eluted directly by boiling in Laemmli buffer. Samples weresubjected to immunoblot analysis, together with inputs.
Subcellular Fractionation. Cytoplasmic and nuclear extracts wereprepared from cells transfected with 1 μg pFLAG-CMV5a orpFLAG-CMV5a-anhydrin using a standard kit (Active Motif)as per manufacturer’s instructions.
FRET Measurements. Image processing software was developed inIDL 6.0 (ITT Visual Information Solutions) to correct the FRETsignal for cross-talk (donor emission into the acceptor channel)and cross-excitation (direct excitation of acceptor molecules atthe donor excitation wavelength) (software available at http://laser.ceb.cam.ac.uk). We used a FRET protocol that is capableof distinguishing the fraction of interacting versus noninteractingmolecules which eliminates artefacts arising from changing inter-action stoichiometries, a method described in detail in a previouspublication (7). Two quantities are evaluated: aFRETand dFRET,which signify the FRETefficiencies, normalized by the fraction ofinteracting acceptors and donors, respectively, from which infor-mation on the interaction stoichiometries can be inferred. Forexample, for dFRET the actual quantity calculated in each imagepixel was
Chakrabortee et al. www.pnas.org/cgi/doi/10.1073/pnas.1013560107 1 of 6
dFRET ¼ N�D
N�D þ ND
E ¼ χDE
which represents a FRET efficiency E normalized by fraction ofinteracting donor molecules. E represents actual FRET transferefficiency between two interacting fluorophores and χD is thefraction of interacting fluorophores (N�
D) divided by the totalnumber of donor fluorophores present in the sample (N�
DþND). A similar expression holds for aFRET. The protocol forFRETanalysis (7) relies on the use of a positive control constructthat yields reproducible and quantifiable levels of FRET. Here a7-amino acid linker with eGFP and mCherry at each end (8) thatyields reproducible FRET efficiencies of E ¼ 15.2� 0.3% wasused. A measurement of the absolute FRET transfer efficiencyfor the linker construct was performed using fluorescence life-time imaging microscopy on a home-built confocal microscopewith time correlated single photon counting (TCSPC) instrumen-tation (9). Empty GFP and mCherry plasmids coexpressed inT-REx293 cells gave insignificant FRET levels (0.2� 0.1%)and were used as negative controls to eliminate contaminatingsignals stemming from a) direct excitation of the acceptor atthe donor excitation wavelength (acceptor cross-excitation),and b) emission of donor fluorescence into the acceptor emissionwindow (donor bleed through). All experiments reported wererepeated three times; per experiment 10 to 12 cells were ana-lyzed. Excitation wavelengths were 488 nm for GFP (donor ex-citation) and 543 nm for mCherry (acceptor excitation).
DNA Binding/Nuclease Assay. His6-tagged AavLEA1 (4), BSA(Sigma) and core histones (Active Motif) were used as controls.For modification of histidine residues, anhydrin was pretreatedwith 100 molar excess of DEPC for 90 min at RT and dialyzedextensively against water for a period of 24 h before use. To ana-
lyze the specific activity of anhydrin, 1 μg of pcDNA3.1 was in-cubated with 2 μg of anhydrin for various time periods at 37 °C.The SC band for each sample was quantitated using ImageJ soft-ware (http://rsbweb.nih.gov/ij/) which gave the mean value of theSC band of each sample. The experiment was repeated threetimes and the average of the mean values was plotted with itsstandard deviation. Conditions for DNA modifying enzymesare (Fig. 6A): lane 2, 2 μg of anhydrin; 5 min, 25 °C; lane 3,DNAse I (2 mU; 4 min, RT); lane 4, micrococcal nuclease(0,2 mU; 4 min, RT); lane 5, T7 endonuclease I (5 U; 15min, 37 °C); lane 6, topoisomerase I (5 U; 15 min, 37 °C); and lane7, DNA gyrase (5 U; 15 min, 37 °C).
Slot-Blot Analysis. For slot-blot analysis, 5 μg anhydrin was incu-bated with 500 ng pcDNA3.1 in quadruplet and samples wereelectrophoresed in 0.6% low melting point agarose gel at 4 °C.OC, linear and SC bands were cut and digested with β-agarase(New England Biolabs) for 1 h at 42 °C. As control, OC andSC forms of nontreated pcDNA3.1 were treated likewise. Samplevolume was reduced in a vacuum concentrator (Eppendorf) andsamples were blotted onto nitrocellulose membrane in a slot-blotapparatus (Fisher Scientific, UK). Membrane was probed withM2 anti-Flag antibody (1∶2;000, Sigma) with purified anhydrin(0.765 μg) as control.
Statistics. One way Anova and Tukey post test using InStat3(GraphPad Software) were performed for CS aggregation andactivity assays as previously described (4). For statistical relevanceof the effect of anhydrin on nuclear aggregation of PABPN1-A17,odds ratio analyses were performed as previously described (4)and in Fig. S3.
1. Uversky V, Gillespie J, & Fink A (2000) Why are “natively unfolded” proteins unstruc-tured under physiologic conditions? Proteins 41:415–427.
2. Browne JA et al. (2004) Dehydration-specific induction of hydrophilic protein genes inthe anhydrobiotic nematode Aphelenchus avenae. Eukaryot Cell 3:966–975.
3. Goyal K et al. (2003) Transition from natively unfolded to folded state induced bydesiccation in an anhydrobiotic nematode protein. J Biol Chem 278:12977–12984.
4. Chakrabortee S et al. (2007) Hydrophilic protein associated with desiccation toleranceexhibits broad protein stabilization function. Proc Natl Acad Sci USA 104:18073–18078.
5. Davies JE, Sarkar S, & Rubinsztein DC (2006) Trehalose reduces aggregate formationand delays pathology in a transgenic mouse model of oculopharyngeal musculardystrophy. Hum Mol Genet 15:23–31.
6. Davies JE, Sarkar S, & Rubinsztein DC (2008) Wild-type PABPN1 is anti-apoptotic andreduces toxicity of the oculopharyngeal muscular dystrophy mutation. Hum MolGenet 17:1097–1108.
7. Elder AD et al. (2009) A quantitative protocol for dynamic measurements of proteininteractions by Forster resonance energy transfer-sensitized fluorescence emission.J R Soc Interface 6:S59–S81.
8. Lleres D, Swift S, & Lamond AI (2007) Detecting protein–protein interactions in vivowith FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). CurrProtoc Cytom Unit 12. 10:1–19.
9. Frank JH et al. (2007) A white light confocal microscope for spectrally resolvedmultidimensional imaging. J Microsc 227:203–215.
10. Berger Z et al. (2006) Deleterious and protective properties of an aggregate-proneprotein with a polyalanine expansion. Hum Mol Genet 15:453–465.
11. Sarkar S, Davies JE, Huang ZB, Tunnacliffe A, & Rubinsztein DC (2007) Trehalose, anovel mTOR-independent autophagy enhancer, accelerates the clearance of mutanthuntingtin and alpha-synuclein. J Biol Chem 282:5641–5652.
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B
C
Log
MW
3
4
5
6
0.2 0.4 0.6
23
45
6
Anhydrin
(Ve-Vo)/(Vi-Vo)
D
17
11
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36
72
28
kDa
95130250A
Anhydrin
1 2 3 4 5 7
40 60 80 100Volume (ml)
Side-chain protons
Amide protons W78
H2O
D2O
10 9 8 71H [ppm]
E
6
F
Anhydrin + D2OAnhydrin + MgCl2Anhydrin + MgCl2 + pcDNA3.1 Anhydrin + CaCl2Anhydrin + CaCl2 + pcDNA3.1
1710 1650 1590Wavenumber (cm-1)
Fig. S1. Purification and structural analysis of recombinant anhydrin. (A) Coomassie-stained SDS/PAGE with size markers (left) and His6-anhydrin-Flag (right).(B) Size exclusion chromatogram of recombinant anhydrin. (C and D) Size exclusion chromatography in a Superdex S75 column shows that anhydrin elutes inthe same fraction as expected for a 26 kDa folded protein. Molecular weight markers are: (1), blue dextran 2,000 (Vo, excluded volume); (2), BSA dimer(134 kDa); (3), BSA monomer (67 kDa); (4), ovalbumin (43 kDa); (5), chymotrypsinogen (25 kDa); (6), ribonuclease A (13.7 kDa); (7), included volume (Vi).Ve is the volume of the protein species. (E) FTIR spectra for anhydrin in D2O (black), in the presence of 10 mM MgCl2 (red) or 10 mM MgCl2 and pcDNA3.1(blue) or 10mMCaCl2 (green) or 10mMCaCl2 and pcDNA3.1 (gray) (25∶1 anhydrin:DNAmolar ratio). (F) Top, 1D 1H-NMR of anhydrin in buffer at 25 °C showingpoor dispersion of amide protons in the region 7.8–8.4 ppm. Bottom, 1D 1H-NMR of anhydrin in D2O, 20 min after dissolution of lyophilized protein.
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0
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AavLEA1 Anhydrin Rnase A Apo-cytochrome C
-synuclein -synuclein BSA -casein
Desiccated + Protein of interest
ControlDesiccated
CS
agg
rega
tion
(a.u
.)
Fig. S2. Citrate synthase (CS) aggregation assay in the presence of various proteins. Aggregation of 0.12 mg CS after two rounds of drying in the absence(black bars) or presence of 10 molar excess of various proteins as marked on the x-axis (gray bars); a nondried control is also shown (white bar).
A B
C
mCherry Anhydrin-mCherry
***
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DAPIEGFP-
PABPN1-A17
Anhydrin-mCherry
DAPIEGFP-
PABPN1-A17
Merge Merge
20µm20µm
Fig. S3. (A and B) Logistic regression analysis of in vivo aggregation assays depicted in Fig. 4J and K. mCherry cells are taken as the control. Black bars representcells expressing both EGFP-PABPN1-A17 and mCherry and gray bars represent cells expressing both EGFP-PABPN1-A17 and anhydrin mCherry. *** denotesp < 0.0001 by logistic regression analysis (estimated odds ratio and 95% confidence intervals). Pooled estimates for the changes in aggregate formationresulting from perturbations assessed in multiple experiments were calculated as odds ratios with 95% confidence intervals. Odds ratio of aggregation =(percentage of cells expressing construct with aggregates in perturbation conditions/percentage of cells expressing construct without aggregates in perturba-tion conditions)/(percentage of cells expressing construct with aggregates in control conditions/percentage of cells expressing construct without aggregates incontrol conditions)]. Odds ratios were considered to be the most appropriate summary statistic for reporting multiple independent replicate experiments ofthis type because the percentage of cells with aggregates under specified conditions can vary between experiments on different days, whereas the relativechange in the proportion of cells with aggregates induced by an experimental perturbation is expected to be more consistent. This method has been usedpreviously (4, 10, 11) to allow analysis of data from multiple independent experiments. Odds ratios and p values were determined using SPSS 9 software (SPSS,Chicago). (C and D) Confocal microscopy of T-REx293 cells after cotransfection with EGFP-PABPN1-A17 expressing construct and (C) mCherry or (D) anhydrin-mCherry in the presence of 10 μM lactacystin. EGFP-PABPN1-A17 expressing nuclei are green (indicative aggregates marked with white arrows; normal nuclearspeckles marked with yellow arrowhead), mCherry and or anhydrin-mCherry expressing cells (or nuclei) are red and the nuclei are stained blue with DAPI.Scale bar: 20 μm.
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Tubulin
His-Anhydrin-Flag
WB:His
WB:Flag
WB:Tubulin
+–His-Anhydrin-Flag
His-Anhydrin-Flag
D
HA-PABPN1-A17
EGFP-PABPN1-A17
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pty
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-PA
BP
N1-
A17
+
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IP: HA WB: EGFP
InputWB: EGFP EGFP-PABPN1-A17
Em
pty
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+
H
is-A
nhyd
rin-F
lag
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HA-PABPN1-A17
No signal
InputWB: HA
IP: HA WB: Flag
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-PA
BP
N1-
A17
+
His
-Anh
ydrin
-Fla
gC
InputWB: Flag
*
A
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His
-Anh
ydrin
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A17
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pty
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His-Anhydrin-Flag
*
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B
Fig. S4. (A) His6-anhydrin-Flag expressed in T-REx293 cells is full length. Total protein extract from untransfected T-REx293 cells or cells transfected with His6-anhydrin-Flag were immunoblotted by probing with antibodies against His6 (top) for N-terminal His-tag, Flag (middle) for C-terminal Flag-tag and also withtubulin as a control (bottom). (B–D) Immunoprecipitation (IP) after expression of (B) empty pCMVFLAG5a or His6-anhydrin-Flag and HA-PABPN1-A17, (C) pHM6vector (empty HA) or HA-PABPN1-A17 with His6-anhydrin-Flag, and (D) pHM6 vector (empty HA) or HA-PABPN1-A17 with EGFP-PABPN1-A17. The inputs fromthe total cell lysates were probed with antibodies against HA (B bottom, C and Dmiddle), Flag (C, bottom) or GFP (D, bottom). IP was performed either (B) withanti-Flag-M2 affinity gel followed by immunoblotting with anti-HA or anti-Flag antibody (B top and middle); or with anti-HA antibody (C and D) followed byimmunoblotting with anti-Flag (C top) and anti-EGFP (D top) antibodies, respectively. The asterisk in (B) and (C) shows the expected position of any HA and Flagsignal, respectively. Gray arrows indicate background bands.
Chakrabortee et al. www.pnas.org/cgi/doi/10.1073/pnas.1013560107 5 of 6
CpcDNA3.1 pHM6 pET28a+
+ + +– Anhydrin––
OC
SCLinear
+AavLEA1
(–) no protein
FBuffer Ca2+ Mg2+ Mn2+
OC
SC
Linear
OC
SC
OC
SC
+ Anhydrin
Anh
ydrin
Hin
dIII
Mar
ker
Bam
HI B
OC
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–
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E25 37 42 55RT
Temp (ºC)
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SC
(–) no protein
+ Anhydrin
A
– 1 5 3015 Control
Time (min)
Anhydrin
OC
SCLinear
HAnhydrin
10
– + + –++10421 ATP (mM)
+– –
OC
SCLinear
– 1 5 3015 60
Time (min)
OC
SC
Linear
100
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60
40
0
20
*** ****** *** ***
OCband
SCband
Anhydrin + pcDNA3.1 + CaCl2
OCband
SCband
pcDNA3.1 + CaCl2
D
G
I
– DEPC++– + Anhydrin
–
Fig. S5. Anhydrin is an endonuclease. (A) Anhydrin-digested pcDNA3.1 compared to plasmid linearized with HindIII or BamHI. (B) Effect of anhydrin onvarious plasmids. (C) Activity of anhydrin pretreated with DEPC. (D, top) pcDNA3.1 (250 ng) after incubation with 2.5 μg anhydrin for various times, and(D, bottom) corresponding polyacrylamide gel showing coomassie-stained anhydrin from the same incubation. The control lane represents an incubationwithout DNA. (E) Effect of temperature on pcDNA3.1 treated with anhydrin (top) or untreated pcDNA3.1 (bottom); in the latter case the plasmid remainedundigested. (F) Cation specificity (Ca2þ∕Mg2þ∕Mn2þ ions) for the endonuclease activity of anhydrin (top). As control, pcDNA3.1 alone (middle) or in thepresence of His6-AavLEA1 (bottom) was incubated with the buffer or various cations and in all these conditions the profile of the plasmid did not change.(G) Slot-blot analysis of pcDNA3.1 after incubation with anhydrin (or in its absence) in the presence of CaCl2 probed with Flag antibody. (H) Effect of addition ofATP. (I) Determination of specific activity of anhydrin. Here 1 μg pcDNA3.1 was incubated with 2 μg anhydrin for various time periods at 37 °C (top). The SC bandfor each sample was quantitated using ImageJ software. The experiment was repeated three times and the average of the values of the quantitated SC bandderived from the software was plotted along with its standard deviation (bottom). ***, p < 0.001. In the above, 200 ng pcDNA3.1 and 2 μg anhydrin were used,unless otherwise stated. Marker sizes are labeled (bp). OC, open circular DNA; SC, supercoiled DNA.
Chakrabortee et al. www.pnas.org/cgi/doi/10.1073/pnas.1013560107 6 of 6
