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Pnas.201413825SI.Pdf Supporting Information Impens et al. 10.1073/pnas.1413825111 13 15 13 15 SI Methods beling) (Silantes Gmbh), or C6 N2 L-lysine HCl and C6 N4 L- Plasmids. pSG5-His6-SUMO1 plasmid encodes the N-terminal arginine HCl (heavy labeling) (Silantes Gmbh). L-Lysine HCl was His6-tagged mature Small ubiquitin modifier 1 (SUMO1) isoform added at its normal concentration in DMEM (146 mg/L), but the (kind gift of A. Dejean, Institut Pasteur, Paris). The pSG5-His6- concentration of L-arginine HCl was reduced to 25 mg/L (30% of SUMO1 T95R mutat was derived from this plasmid using PCR the normal concentration in DMEM) to prevent metabolic con- mutagenesis. pSG5-His6-SUMO2 was obtained by inserting the version of arginine to proline (4). Cells were kept for at least six cDNA corresponding to the human mature SUMO2 isoform population doublings to ensure complete incorporation of the la- with an N-terminal His6 tag in the pSG5 vector (Stratagene). beled lysine and arginine. 2 The pSG5-His6-SUMO2 T91R mutant was derived from this For transfections, cells were seeded in 75-cm flasks or in 6- or plasmid by PCR mutagenesis. N-terminally HA-tagged human 24-well plates at a density of 2.7 × 106 cells per flask or 3 × 105 or cDNA of ZBTB20 (Zinc finger and BTB domain containing 0.5 × 105 cells per well, respectively. The next day cells were 20) isoform 2 (UniProt identifier Q9HC78-2), HMBOX1 (Ho- transfected with Lipofectamine LTX reagents (Invitrogen) (20 μg meobox containing protein 1) isoform 1 (HMBOX1A) (UniProt of DNA per flask, 3.5 μg per well in the six-well plates, or 0.75 μg identifier Q6NT76-1), NACC1 (Nucleus accumbens-associated per well in the 24-well plates) for 48 h. protein 1) (UniProt identifier Q96RE7), MAP7 (Microtubule-as- For listeriolysin O (LLO) treatment, cells were serum-starved sociated protein 7) isoform 1 (UniProt identifier Q14244-1), or for 2 h; then 3 nM of purified toxin (5) was added directly to the LMNB1 (Lamin-B1) (UniProt identifier P20700) and C-terminally culture medium for 20 min. Cells were lysed further for His HA-tagged human cDNA of TFAP2A (Transcription factor AP-2 pulldown assays or immunoblot analysis. alpha) isoform 1 (UniProt identifier P05549-1) were introduced into the pCDNA3 vector (Invitrogen). From these plasmids ex- Luciferase Assays. HeLa cells were transfected with different mixes pression vectors for HA-ZBTB20 K257R, HA-HMBOX1 K413R, of NRLuc- and CRLuc-encoding plasmids (ratio 1:1). Cells were HA-NACC1 K167R, HA-MAP7 K406R, HA-LMNB1 K241R, harvested 48 h after transfection, and luciferase activities were and TFAP2A K10R-HA were derived by PCR mutagenesis. The quantified on a Tristar LB491 luminometer (Berthold Technol- pCDNA3-NRLuc plasmid was obtained by inserting the coding ogies), using the Renilla Luciferase Assay System (Promega). sequence corresponding to N-terminal residues 1–229 from the green Renilla luciferase protein (RLuc; ThermoFisher Scientific), His Pulldown Assays. SUMOylated proteins were isolated from cell fused to a GGGS flexible linker, into pCDNA3 vector (1). From lysates as described in ref. 6. Briefly, cells were washed in PBS this plasmid four expression vectors were derived with SUMO1 and lysed in lysis buffer [6 M guanidium HCl, 10 mM Tris, 100 WT, SUMO1 T95R, SUMO2 WT, or SUMO2 T91R fused in the mM sodium phosphate buffer (pH 8.0), 5 mM β-mercaptoetha- C-terminal luciferase fragment (residues 1–229). The pCDNA3- nol, 1 mM imidazole]. For MS analysis, 2 × 107 cells per SILAC CRLuc plasmid was obtained by inserting the coding sequence condition were lysed in 40 mL of lysis buffer without β-mer- corresponding to the C-terminal residues 230–311 from the RLuc captoethanol and imidazole. Equal volumes of each SILAC protein, fused to three GGGS flexible linkers, into the pCDNA3 condition were mixed, and proteins were reduced and alkylated vector (1). Expression vectors were derived from this vector for by the addition of 5 mM Tris(2-carboxyethyl)phosphine (TCEP) either a phosphorylation-mimic variant of the Daxx (Death do- and 10 mM chloroacetamide. After incubation for 30 min at 37 °C main-associated protein) SUMO-interacting motif (SIM) (KGG- in the dark, excess chloroacetamide was quenched by addition of KTSVATQCDPEEIIVLDDDD) (1) or the PIAS2 (Protein 20 mM DTT. Then cell lysates were incubated overnight at 4 °C inhibitor of activated STAT) SIM (KVDVIDLTIESSSDEEEDP- with 3 mL of packed NiNTA agarose beads (Qiagen) prewashed PAKR) (2), fused in the N terminus of the luciferase fragment in lysis buffer. After incubation, beads were washed once in lysis – (residues 230 311). All generated constructs were verified by buffer, once in wash buffer pH 8.0 [8 M urea, 10 mM Tris, 100 sequencing. mM sodium phosphate buffer (pH 8.0), 0.1% Triton X-100, 5 mM β-mercaptoethanol], and three times in wash buffer pH 6.3 Antibodies. The following primary antibodies were used for im- [8 M urea, 10 mM Tris, 100 mM sodium phosphate buffer (pH munoblot analysis: mouse anti-actin (R5441; Sigma-Aldrich) and β anti-HA tag (no. 2367; Cell Signaling Technology); rabbit anti- 6.3), 0.1% Triton X-100, 5 mM -mercaptoethanol, 10 mM im- RanGAP1 (no. R0155; Sigma-Aldrich) and anti-SUMO1 (no. 4930; idazole]. SUMOylated proteins then were eluted from the beads Cell Signaling Technology); and home-made rabbit polyclonal using elution buffer A [200 mM imidazole, 5% (wt/vol) SDS, 150 · β antibodies raised against recombinant SUMO3 protein produced in mM Tris HCl (pH 6.7), 30% (vol/vol) glycerol, 720 mM -mer- Escherichia coli (R206). Anti-mouse and anti-rabbit HRP-conju- captoethanol, and 0.0025% bromophenol blue] for immunoblot gated antibodies (AbCys) were used as secondary antibodies. analysis or elution buffer B [100 mM sodium phosphate buffer (pH 6.8), 200 mM imidazole] for MS analysis. The latter eluates Cell Culture, Stable Isotope Labeling by Amino Acids in Cell Culture, contained about 500 μg of protein in a volume of 1.5 mL for both and Transfections. HeLa cells (CCL-2; ATCC) were cultured in SUMO1 and SUMO2 analysis. MEM-GlutaMAX medium (Invitrogen), supplemented with 10% (vol/vol) FBS, MEM nonessential amino acids (Invitrogen), and Immunoblotting. Cells lysed in Laemmli buffer [0.125 M Tris (pH 1 mM sodium pyruvate (Invitrogen). 6.8), 4% (wt/vol) SDS, 20% (vol/vol) glycerol, 100 mM DTT] and For stable isotope labeling by amino acids in cell culture (SILAC) proteins eluted from His pulldown assays were separated on SDS- (3), cells were cultured in DMEM without L-lysine, L-arginine, or polyacrylamide gels. Proteins were transferred to PVDF mem- L-glutamine (Silantes Gmbh) supplemented with 10% (vol/vol) branes and incubated with primary and secondary antibodies. dialyzed FBS (Invitrogen), 2 mM GlutaMAX (Invitrogen), and Proteins were revealed using Pierce ECL 2 Western Blotting either natural L-arginine HCl and L-lysine HCl (light labeling) Substrate (Fisher Scientific). All displayed immunoblots are 13 (Sigma), D4 L-lysine HCl and C6 L-arginine HCl (medium la- representative of at least two independent experiments. Impens et al. www.pnas.org/cgi/content/short/1413825111 1of5 Immunocapture of Diglycine-Modified Peptides. Eluates from the cation of lysine residues (+114.042927 Da; light search), SILAC His pulldown were diluted further with 8.5 mL 50 mM ammonium modification of lysine residues, and GG modification of SILAC- bicarbonate, and proteins were digested with 20 μg trypsin labeled lysine residues (+4.025107 Da and +118.068034 Da, (Promega). Immunocapture of diglycine (GG)-modified pep- medium search; +8.014199 Da and +122.057126 Da, heavy tides then was performed using the PTMScan Ubiquitin Rem- search). SILAC modification of arginine residues was set as an nant Motif (K-e-GG) Kit (Cell Signaling Technology) according additional fixed modification for the medium (+6.020129 Da) to the manufacturer’s instructions. Briefly, peptides were puri- and heavy (+10.008269 Da) searches. For all searches mass fied on Sep-Pak C18 cartridges (Waters), lyophilized for 2 d, and tolerance of the precursor ions was set to 10 ppm, and mass redissolved in 1.4 mL of the 1× immunoprecipitation buffer tolerance of the fragment ions was set to 0.5 Da. The peptide without detergent supplied with the kit. Note that at this point an charge was set to 2+,3+, and 4+, and up to three missed tryptic aliquot corresponding to 4 μg of digested protein material was cleavage sites were allowed. Also, the C13 setting of Mascot was taken to analyze the input. Peptides were incubated with the set to 1. To identify the phosphorylated peptides listed in Da- antibody-bead slurry for 2 h on a rotator at 4 °C, and after taset S3, these searches were repeated with phosphorylation of μ several wash steps GG-modified peptides were eluted in 100 L serine, threonine, and tyrosine residues as additional variable 0.15% TFA and desalted on reversed-phase C18 OMIX tips modifications. Only peptides that were ranked first and scored ’ (Agilent) according to the manufacturer s protocol. Purified above the threshold score set at 99% confidence were withheld. GG-modified peptides were dried under vacuum in HPLC in- For processing of all MS data the ms_lims (version 7.7.7) soft- − serts and stored at 20 °C until LC-MS/MS analysis. ware platform was used (8). In total, 2,016 and 3,123 peptide spectrum matches (PSMs) were obtained for the SUMO1 and LC-MS/MS Analysis. Peptides were redissolved in 20 μL of solvent A SUMO2 analysis, respectively, with a false-discovery rate (FDR) [0.1% formic acid in water/acetonitrile (98:2, vol/vol)], of which <0.3% (9).
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