Cst2764

Proteins were separated by SDS–PAGE (Mini-Protean TGX Precast Gel 12%, 456-1045, Bio-Rad) and transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Membranes were blocked with dry milk dissolved in Tris buffer saline with 1% Tween 20 (TBST) for 1 h and probed overnight at 4 °C with the primary antibodies of interest directed against: rabbit anti-BCL-2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-BID (CST2002, Cell Signaling), rabbit anti-BAK (CST12105, Cell Signaling), rabbit anti-BAX (CST2772, Cell Signaling), rabbit anti-Actin (CST4970, Cell Signaling) followed by anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling) in 3% BSA in TBST for 1 h at room temperature. Immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad). When necessary, immunoblots were stripped in 0.1 M glycine pH 2.5, 2% SDS for 40 min and washed in TBS. Bands were visualized with LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to measure the integrated optical density of bands.

Proteins were separated by SDS-PAGE (Mini-Protean TGX Precast Gel 12%, 456-1045, Bio-Rad, Madrid, Spain) and transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Membranes were blocked with dry milk dissolved in Tris-buffered saline with 1% Tween 20 (TBST) for 1 h and probed overnight at 4 °C with the primary antibodies of interest directed against rabbit anti-BCL-xL (CST2764, Cell Signaling, Leiden, The Netherlands), rabbit anti-MCL-1 (CST5453, Cell Signaling, Leiden, The Netherlands), rabbit anti-BIM (CST2933, Cell Signaling, Leiden, The Netherlands), rabbit anti-actin (CST4970, Cell Signaling, Leiden, The Netherlands) followed by anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling, Leiden, The Netherlands) in 3% BSA in TBST for 1 h at room temperature. Immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad, Madrid, Spain). When necessary, immunoblots were stripped in 0.1 M glycine pH 2.5, 2% SDS for 40 min, and washed in TBS. Bands were visualized with LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ were then used to measure the integrated optical density of bands.