Mouse anti bcl 2 clone 10c4

After washing with PBS, 3 × 10⁶ cells were directly lysed in pre-heated H8 buffer (20 mM Tris/HCl pH 7.5, 2 mM EGTA, 2 mM EDTA, 1% SDS, supplemented with 50 mM DTT) and boiled and homogenized at 95 °C in the presence of 4× Lämmli buffer. Proteins were separated on 12.5% or 15% denaturing SDS-PAGE gels and transferred to polyvinilydene difluoride (PVDF) membrane (Immobilon-FL, 0.45 μM, Merck Millipore, Zug, CH). After blocking, the membranes were probed overnight with the following primary antibodies: mouse anti-BCL-2 (clone 10C4, BioLegend); rat anti-BIM (clone 3C5) and rat anti-MCL-1 (clone 14C11), kind gifts from D. Huang (Parkville, Australia); mouse anti-GSK-3α/β (clone 0011-A/ 1H8) form Santa Cruz Biotechnology (Dellas, TX, US); rabbit anti-phospho-AKT (Ser473) (clone D9E) and mouse anti-AKT (clone 40D4) from Cell Signaling Technology (Danvers, MA, US); mouse anti-tubulin (clone B-5-1-2) from Sigma; mouse anti-GAPDH from Merck Millipore (Zug, CH); mouse anti-actin from BD Biosciences (San Jose, CA, US). For all immunoblots with total lysates, near-infrared fluorophore-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, DE) were used. All immunoblots were analyzed and quantified with the Odyssey^® Fc Dual-Mode Imaging System using the ImageStudio software 3.1.4 (LI-COR).