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).
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).