Proteins were separated and detected as previously described [28 (link)]. In brief, SDS-PAGE gel (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) was used to separate proteins and then transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Blocking of membranes was achieved by using 5% dry milk dissolved in Tris Buffer Saline with 1% Tween 20 (TBST) and the following antibodies were incubated overnight at 4 °C: rabbit anti-BCL-2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit anti-NOXA (CST14766, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-phospho-ERK1/2 (CST4376, Cell Signaling), rabbit anti-Actin (CST4970, Cell Signaling). Anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling) was used and immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad). When required, immunoblots were stripped in 0.1 M glycine pH 2,5, 2% SDS for 40 min and washed in TBS. The visualization of the bands was done using the LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to quantify the integrated optical density of bands.
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