Iblot gel transfer stacks pvdf membranes

Cell homogenates were obtained in RIPA buffer (Pierce, Thermo Scientific), supplemented with 1 × EDTA-free complete protease inhibitor cocktail (Roche). Protein concentration was quantified by a modified Lowry assay (DC protein assay; Bio-Rad). 30 μg of protein were resolved in NuPAGE 4-12% Bis-Tris gels and transferred to iBlot Gel Transfer Stacks PVDF membranes (Life Technologies, Thermo Fisher Scientific). After blocking with Tris-buffered saline with Tween-20 containing 5% non-fat dry milk or 5% BSA for 1 h at room temperature, membranes were probed overnight at 4°C with the following antibodies: anti-PARP [1:2000, Cell Signaling #9542]; anti-CDC25A [1:1000, Santa Cruz #sc-7389]; anti-WEE1 [1:1000, Santa Cruz #sc-5285]; anti-CHK1 [1:2000, Santa Cruz #sc-8408]; anti-BCL2 [1:1000, Dako #M0887]; anti-AKT3 [1:1000, Upstate #05-780]; anti-VEGF [1:1000, Abcam #ab46154]; anti-Mouse IgG-Peroxidase antibody produced in rabbit (1:10.000, Sigma-Aldrich #A9044); anti-Rabbit IgG-Peroxidase antibody produced in goat (1:10.000, Sigma-Aldrich #A0545) and anti-Actin HRP [1:40.000, Santa Cruz #sc-1616]. Membranes were developed with SuperSignal Dura detection kit (Pierce) or EZ-ECL Chemiluminescence detection kit (Biological Industries, Kibbutz Beit-Haemek, Israel).

Cell lysates were obtained in RIPA buffer (Pierce, Thermo Scientific), supplemented with 1x EDTA-free complete protease inhibitor cocktail (Roche). Protein concentration was quantified by a modified Lowry assay (DC protein assay; Bio-Rad). 30 μg of protein were resolved in NuPAGE 4–12% Bis-Tris gels and transferred to iBlot^® Gel Transfer Stacks PVDF membranes (Life Technologies, Thermo Fisher Scientific). After blocking with Tris-buffered saline with Tween-20 containing 5% non-fat dry milk or 5% BSA for 1 h at room temperature, membranes were probed overnight at 4°C with the following antibodies: anti-KLF4 [1:1000, Santa Cruz #sc-20691]; anti-Rabbit IgG-Peroxidase antibody produced in goat [1:10.000, Sigma-Aldrich #A0545] and anti-Actin HRP [1:40.000, Santa Cruz #sc-1616]. Membranes were developed with SuperSignal Dura detection kit (Pierce) or EZ-ECL Chemiluminiscence detection kit (Biological Industries, Kibbutz Beit-Haemek, Israel).