Antibodies against PML (PG-M3, sc-966, H-238, and sc-5621), p53 (DO-1, sc-126), Ubc9 (sc-10759) and Mdm2 (SMP14, sc-965), and horse radish peroxidase (HRP)-conjugated anti-rabbit, anti-mouse, and anti-goat IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p53 (Ab-6) and Mdm2 (Ab-1) were purchased from EMD Chemicals (Gibbstown, NJ), TRIM27 antibody (18791) from Immuno-Biological Lab (Minneapolis, MN), and Flag peptides and anti-Flag antibody (M2) from Sigma (St. Louis, MO). Glutathione Sepharose 4B (17-0756-01) (GE healthcare); SUMO E1, Ubc9, SUMO1, SUMO2 and ATP (Boston Biochem, Boston, MA); and MG132 (BML-PI102) (Enzo life science, Plymouth Meeting, PA) were purchased from the indicated sources.
Plasmids for expressing PML (isoform IV), TRIM27, TRIM32, TRIM5δ, Mdm2, c-Jun, IκBα, SUMO1, and SUMO2 in mammalian cells and/or in the in vitro translation system were constructed in pRK5 with an N-terminally fused Flag, HA, or 6xHis (His) tag, or glutathione-S-transferase (GST) as indicated. Other TRIM plasmids were kindly provided by Drs. V. Yu, A.-M. Herr, F. Rauscher, III, and T. C. Cox. TRIM25 was purchased from Addgene (Cambridge, MA). For expressing proteins in Escherichia coli, full-length PML and TRIM27 were fused to GST in pGEX-1ZT, a derivative of pGEX-1λT. p53 was fused to the 6xHis tag at the C-terminus and the Flag tag at the N-terminus in pET28a. Point mutations were made by overlap PCR. All plasmids generated for this study were confirmed by sequencing.
In vivo SUMOylation assays
SUMOylation assays were performed as described previously with minor changes (Chen and Chen, 2003
). Cells cultured in 6-cm plates were transfected with His-SUMO1 or His-SUMO2 expression plasmids, MDM2, GFP, and TRIM protein expression plasmids. 24 h after transfection, cells were treated with MG132 (20 nM) for 4 h. Cells from each plate were collected into two aliquots. One aliquot was lysed in lysis buffer and analyzed by western blot to examine the expression of transfected proteins. The second aliquot was lysed in buffer A (6 M guanidinium-HCl, 0.1 M Na2
, 10 mM Tris-Cl pH 8.0, 5 mM imidazole, and 10 mM β-mercaptoethanol), and incubated with Ni2+
-NTA beads (Qiagen) for 4 h at room temperature or overnight at 4 °C. The beads were washed sequentially with buffers A, B (8 M urea, 0.1 M Na2
, 10 mM Tris-HCl pH 8.0, 10 mM β-mercaptoethanol), and C (same as B except pH=6.3). Beads with bound proteins were then boiled in SDS sample buffer, the proteins were fractioned by SDS–PAGE and were analyzed by western blot for the presence of conjugated Mdm2, p53 or PML.
Protein generation and purification
Flag-tagged PML, its mutants, and TRIM27 were expressed in Pml−/−
MEFs or 293T cells through transient transfection and purified by anti-Flag M2 beads as described (Tang et al., 2006
; Tang et al., 2004
). Whole cell lysates were made in IP-lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM PMSF, 1 mM DTT, plus EDTA-free “complete” protease inhibitors). The lysates were immunoprecipitated with the anti-Flag M2 beads for 4 h to overnight. After extensive wash, the proteins were eluted from beads with 100 μg/ml 3xFlag peptide in elution buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM DTT, 25 μM ZnCl2
, and 10% glycerol).
GST-fusion proteins and His-p53/pET28a were induced and purified in Escherichia coli BL21(DE3). Escherichia coli BL21(DE3) expressing the appropriate GST-fusion proteins was cultured in Terrific Broth (TB) medium with 50 μM ZnCl2. The proteins were induced for 4 h with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG), followed by centrifugation, and re-suspended in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 5 mM DTT, 25 μM ZnCl2 plus EDTA- free “complete” protease inhibitors). Cellswere sonicated and then centrifuged for 10 min at 4 °C. Extracts were incubatedwith 30 μl glutathione Sepharose 4B beads (50% slurry) for 4 h at 4 °C. After extensive washing, the beads with the GST-fusion proteins were used for in vitro SUMOylation assay. BL21 (DE3) containing His-p53/pET28a was cultured in TB medium without ZnCl2. His-p53 was captured with TALON His-Tag purification resins (Clontech, Mountain View, CA). After extensive washing, His-p53 was eluted from the TALON beads by 1x PBS buffer containing 300 mM imidazole. His-p53 was then passed through a PD-10 desalting column (GE Healthcare) pre-equilibrated with a buffer containing 20 mM Tris-HCl, pH8.0, 150 mM NaCl, 0.1 mM EDTA, and 5% glycerol.
In vitro translation
In vitro-translated proteins were made using TNT® Coupled Transcription/Translation System with S6 RNA polymerase (Promega).
In vitro SUMOylation assays
In vitro SUMOylation reactions were performed at 37 °C for 1 h in 20 μl volume containing purified His-p53 (~100 ng) or in vitro translated HA-Mdm2 (4 μl), mammalian or bacterial cell-expressed PML or TRIM27 protein (5–10 ng), SAE1/SAE2 (125 nM), Ubc9 (50 nM), and His-SUMO1 or His-SUMO2 (32 μM). The reaction buffer contained 50 mM Tris-HCl pH 7.5, 2.5 mM Mg2+-ATP, and 2.5 mM DTT. Mdm2-containing reaction mixes were incubated with Ni2+-NTA beads. After wash in Urea buffer, the conjugated Mdm2 was detected by anti-Mdm2 antibody (SMP14, Santa Cruz). p53-containing mix was directly fractionated by SDS-PAGE (8%) and analyzed by western blot using anti-p53 antibody (DO-1).
Cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100, blocked with 1% BSA and incubated with anti-TRIM27, anti-p53, anti-Mdm2, and anti-PML antibodies as indicated, followed by Texas-red conjugated anti-Rabbit IgG (Vector Laboratories, Burlingame, CA) and FITC-conjugated anti-mouse IgG antibody (Zymed Laboratories). The cells were mounted with DAPI-containing medium (Vector Laboratories, Burlingame, CA) and the images were acquired with a confocal microscope.
Whole cell lysates were made in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM PMSF, 1 mM EDTA, 1 mM DTT, plus EDTA-free “complete” protease inhibitors (Roche: 04693132001). The lysates were then immunoprecipitated with the anti-Flag M2 beads for 4 h to overnight. Beads were washed multiple times and boiled in SDS-containing loading buffer. Protein samples were resolved by SDS-PAGE and transferred onto PVDF membrane and probed with the indicated antibodies.
In vitro GST pull down assay
GST-pull down was performed as described previously (Townson et al., 2006
) with some modifications. GST only or GST-fusion proteins were induced in Escherichia coli
BL21. Cellswere lysed in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1mM EDTA, 0.1% SDS, 1 mM PMSF, plus complete protease cocktail) and sonicated. 400 μg of GST or 1 mg of GST-fusion protein bacterial crude extracts was incubated with 30 μl glutathione Sepharose 4B beads (50% slurry). Flag-TRIM27 was purified from 293T cells as described above. For the binding assay, the beads were incubated in lysis buffer and Flag-TRIM27 elute was added and incubated at 4 °C. The beads were washed three times with lysis buffer. Bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and analyzed by western blot.
Protein half-life analysis
HeLa cells transfected with appropriate vectors were cultured for 18 h. Cells were divided into four fractions and further cultured on 60-mm dishes overnight. The cells were treated with 50 μg/ml cycloheximide (CHX) and collected at different time points. Cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM PMSF, plus complete protease cocktail). Proteins were separated by SDS-PAGE and analyzed by Western blots.
Cells were seeded in 6-cm plates and transfected with 80 pmol siRNA per well using Lipofetamine 2000 according to the manufacturer’s instruction. Ubc9 siRNA was ordered from Thermo Scientific Dharmacon (Cat# M-004910-00). Control siRNA was ordered from Qiagen (Cat# 1027281).
In vitro ubiquitination assay
In vitro ubiquitination reactions were performed at 37 °C for 2 h in 20 μl volume containing purified His-p53 (~100 ng) or bacterial-expressed TRIM27 protein (5–10 ng), E1 (156 nM), Ubc5a (50 nM), and Ubiquitin (147μM). The reaction buffer contained 50 mM Tris-HCl pH 7.5, 2.5 mM Mg2+-ATP, and 2.5 mM DTT. p53 ubiquitination was analyzed by Western blot using anti-p53 antibody (DO-1).