Anti mdm2 ab 1

Total cell lysates and immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted by a semi-dry transfer in an electric field onto polyvinylidene difluoride membranes (PVDF; Millipore). After transfer, the membranes were blocked in 5% low fat dry milk in TBST (10 mM TRIS-HCl, 100 nM NaCl and 0.05% Tween 20; pH 7.4) for 1 h at room temperature and incubated overnight at 4°C with a primary antibody in 1% low fat dry milk in TBST. The following primary antibodies were used: anti-TFII-I (V-18; Santa Cruz Biotechnology), anti-GST (GE Healthcare), anti-Mdm2 (Ab-1; Merck-Millipore), anti-Tubulin alpha (Exbio), anti-PCNA, (PC-10, kindly provided by Borivoj Vojtesek, Masaryk Memorial Cancer Institute, Brno, Czech Republic), anti-β-galactosidase (BG-02; Santa Cruz Biotechnology), anti-Myc (9E10, Santa Cruz Biotechnology), anti-Flag (M2; Sigma-Aldrich) and anti-GFP (Roche), using dilutions recommended by the manufacturers. After three washes in 1% milk in TBS, membranes were incubated one hour at room temperature with secondary antibodies conjugated to horseradish peroxidase (GE Healthcare). Enhanced chemiluminescence kit (ECL; GE Healthcare) and blue light sensitive medical X-ray film (Agfa) or G:BOX Chemi imaging system (Syngene) were used to visualize proteins of interest.

U2OS cells in 60-mm dishes were transfected with 0.1 μg FLAG-p53 and 1.4 μg Mdm2 mutant plasmid constructs using Effectene transfection reagent according to guidelines of the manufacturer (Qiagen). Each transfection mixture also contained 0.2 μg pEGFP-N1 (Clontech) to control for transfection efficiency, and empty plasmid pcDNA3.1 (Invitrogen) was used to bring the total amount of transfected DNA to 1.7 μg. Cells were washed with cold PBS 30 h post-transfection and lysed with 0.25 ml of SDS sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with anti-Flag M2 (Sigma-Aldrich, F3165, 1:3,000), anti-Mdm2 Ab-1 (Merck, OP46, 1:1,000), and anti-GFP 7.1/13.1 (Roche, 11814460, 1:5,000) antibodies. In some experiments, p53 was also detected using the anti-p53 monoclonal antibody DO-1 (used at 0.2 μg/ml, kindly provided by Borivoj Vojtesek, Masaryk Memorial Cancer Institute, Brno).

U2OS cells grown in 60-mm dishes were transiently transfected with FLAG-p53 (0.5 μg), hemagglutinin (HA)-ubiquitin (0.5 μg), and Mdm2 plasmid constructs (5 μg) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. Empty plasmid pcDNA3.1 (Invitrogen) was used to bring the total amount of transfected DNA to 6 μg. Cells were treated 24 h post-transfection with 15 μM MG132 for 3 h, washed with PBS, and lysed in 0.3 ml 0.5% SDS. Lysates were boiled for 5 min, vortexed, cooled down to room temperature, and diluted with 1 ml of Triton X-100 lysis buffer. p53 was immunoprecipitated using 1 μg of anti-p53 monoclonal antibody DO-1 on a rotating wheel at 4°C. After 1 h of incubation, 20 μl of protein G-Sepharose beads (Sigma-Aldrich) were added for 45 min. The immunoprecipitates were washed three times with lysis buffer and analyzed by Western blotting. Ubiquitylated p53 was detected using anti-HA-Peroxidase antibody (Roche, 12013819001, 1:2,000); FLAG-p53 and MDM2 levels in the input were detected using anti-FLAG antibody M2 (Sigma-Aldrich), anti-Mdm2 Ab-1 (Merck).