Parp f 2

HCT116 cells were washed with ice-cold PBS and lysed on ice in lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor cocktail (Roche). Cell debris was cleared by centrifugation. Whole cell extracts were resolved by 10% SDS-PAGE, transferred onto nitrocellulose membranes, and blotted with antibodies against Sam68 (07-415, Millipore), p53 (HAF1355, R&D), MDM2 (Ab-5, Millipore), p21 (C-19, Santa Cruz), Bax (P-19, Santa Cruz), Puma (4976, Cell Signaling), PARP (F-2, Santa Cruz) and β-actin (A3853, Sigma Aldrich). For immunoprecipitation, anti-Sam68 (07-415, Millipore), anti-p53 (1C12, Cell Signaling), anti-Myc (05-724, Sigma), anti-GFP (Anti-GFP, Roche), mouse or rabbit IgG (Santa Cruz) antibodies were incubated for 2 h with whole cell lysates, and 1 h with 50 μl of 50% slurry protein A/G beads (Sigma) at 4°C. Beads were washed three times with lysis buffer, and bound proteins were recovered by boiling in Laemmli sample buffer.

Cells treated with DMSO and Ph were harvested for immunoblotting analysis [25 (link)]. Primary antibodies were purchased from multiple sources. Antibodies against WAF1/Cip (p21, #2947), Rb (#9309), cyclin D1 (#2922), cyclin E1 (#4129), phospho-FAK (Tyr397, #3283), FAK (#3285), phospho-Src (Tyr416, #2101) and E-cadherin (#14472) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-Kip1 (p27, # 610241) was purchased from BD Bioscience Pharmingen (San Diego, CA, USA). Anti-GAPDH (ab9485), anti-paxillin (ab32084), anti-alpha smooth muscle actin (α–SMA, ab21027), anti-N-cadherin (ab18203) and anti-Src (ab47405) antibodies were purchased from Abcam (Cambridge, UK). Antibodies against GLUT2 (H-67), p53 (DO-1), and PARP (F2) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).