Smp14

Antibodies against CDK4 (3F121), MDM2 (SMP14), total Rb (IF8), cyclin A (H432), p16 (C20), p53 (DO-1 and Bp53-12), tubulin (C20) and FLAG (M2) were obtained from Santa Cruz Biotechnology, phospho-Rb 780 (#9307) from Cell Signalling, Arf (3642) from Abcam, and ATRX (A301-045A) from Bethyl Laboratories. Treated cells were lysed with buffer composed of 50mM Tris-HCl, pH7.4, 250mM NaCl, 5mM EDTA, 0.5% NP40, 2mM PMSF, and supplemented with protease inhibitors. Forty to eighty micrograms of protein were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight with antibodies.
Extracts were prepared from pre-treatment biopsies within two weeks before the first dose of the drug, and post-treatment biopsies were collected within six days of the start of the second cycle. Extracts were prepared in 50mM Tris-HCl, pH7.4, 150mM NaCl, 1mM EDTA, 1% NP40, 0.25% sodium deoxycholate and supplemented with mini-protease inhibitor cocktail (Roche). Tumor response was assessed by reference radiologist by CT scan every six weeks for 36 weeks, and every 12 weeks thereafter. The clinical trial was approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center and all patients provided written informed consent (NCT01209598).

Total protein extraction was performed using cell lysis buffer (Cell Signaling Technology, Frankfurt, Germany) according to the manufacturer's instructions. The protein concentration in the supernatant was determined with a Bradford assay. Western blotting was performed using 15 μg protein per sample, which were separated by SDS-polyacrylamide gel electrophoresis and transferred on a polyvinylidene fluoride membrane (Carl Roth, Karlsruhe, Germany) by electroblotting. Antibodies were incubated in 5% non-fat milk in TBST (0.1% Tween-20, 20 mM Tris, 140 mM NaCl, pH 7.6) overnight at 4°C. Proteins were detected with antibodies against p53 (DO-7, Dako Deutschland GmbH, Hamburg, Germany), p21 Waf1/Cip1 (12D1), Puma (D30C10), Gadd45a (D17E8), Bax (D2E11), CDK1 (8G10) (Cell Signaling Technology, Danvers MA, United States) and Mdm2 (SMP14, Santa Cruz, Heidelberg, Germany) using the biotechnology SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher, Rockford IL, USA).

GST-MDM2, p21-Luc, pCMV-Bam-MDM2, and MDM2ΔRING have been described previously [20 (link), 21 ]. Myc-Mdm2 was kindly provided by Dr. Jochemsen. All Ub and Ub mutants were PCR-amplified and subcloned into pET28a. The mouse Mdm2 was also cloned into pcDNA3 and confirmed by sequence. Flag-p73α, p73β, and p73 mutants were generated by PCR and subcloned into pCMV-Tag1 (Stratagene). p73α and p73β were also cloned into pcDNA3 without tag. All PCR products were confirmed by sequencing. Anti-p73 (Ab-1 and Ab3, Oncogene Science; H-79, Santa Cruz Biotechnology; ab40658, Abcam), anti-MDM2 (2A10, Calbiochem; SMP14, Santa Cruz Biotechnology; MD-219, Sigma), anti-Myc (9E10, Roche), anti-Flag (M5, Sigma), anti-GFP (B-2, Santa Cruz Biotechnology), anti-GST (B-14, Santa Cruz Biotechnology), anti-HA (12CA5, Roche), anti-ubiquitin (BD Bioscience), anti-actin (Sigma), anti-CD20 (Pharmingen), and polyubiquitin-specific FK-1, anti-ubiquitin, Lys63-specific and Lys48-specific antibodies (Millipore) were used according to the manufacturers’ instructions.

Western blot analysis was performed using 10 µg of whole cell extracts as described [9] (link), with modifications. Proteins were electrophoresed on a 5–20% sodium dodecyl sulfate polyacrylamide gradient gel (Atto). Blots were labeled using primary antibodies against TP53 (1∶1000, Ab-6; Merck Millipore), phospho-TP53-Ser15 (1∶1000; Cell Signaling), p21 (1∶1000; BD Biosciences), MDM2 (1∶200, SMP-14; Santa Cruz Biotech), MDMX/HDMX (1∶5000; Bethyl), CHEK2 (1∶1000; Cell Signaling), phospho-CHEK2-Thr68 (1∶1000; Cell Signaling), β-tubulin (1∶1000; Sigma-aldrich), and β-actin (1∶10,000; Sigma-aldrich).