Hamster anti bcl 2

Cell extracts from sorted Kasumi-1 CD34^+/- fractions were prepared by cell suspending in ice-cold lysis buffer, containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% Triton X, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotonin and 5 μg/ml leupeptin. Pellets were boiled for 10 min, electrophoresed in 10–12% sodium dodecyl sulfate – polyacrylamide gel, transferred to 0.45 μm PVDF membrane and immersed for 1 hour in a blocking solution (0.5% Tween-20, 5% BSA or milk). The membranes were incubated overnight with primary antibodies: mouse-anti-p44/p42 (pT202/pY204; cat#9101 S), rabbit-anti-p42 (cat#9108 S), rabbit-anti-pAKT (ser473; cat# 4060 S), rabbit-anti-AKT (1,2,3; cat#9272) all from Cell Signaling Inc., hamster-anti-BCL-2 (BD pharmingen; cat#51-1513GR) and mouse-anti-GAPDH (Abcam; cat#ab9484). The blots were washed three times with TBST buffer [10 mM Tris (pH 7.4), 100 mM NaCl 0.5%-Tween-20], incubated for 1 hour with secondary antibodies conjugated to horseradish peroxidase and re-washed. Blots were developed using the WesternBright ECL HRP substrate (Advansta Inc, San Jose, CA) in ImageQuant LAS 4000 (GE Healthcare).

Cells were lysed with CHAPS lysis buffer (containing 10 mM HEPES, 150 mM NaCl, 1% CHAPS (Sigma), pH 7.4) supplemented with protease inhibitor cocktail (Roche, 1169749800). To measure conformational changes associated with the activation of BAX and BAK mouse anti-BAX clone 6A7 (Sigma) or mouse anti-BAK AB-1 (Calbiochem) were used as described previously^{29 (link)}. For analysis of interactions within the BCL-2 family, mouse anti-MCL-1 (BD Bioscience, 559027), hamster anti-BCL-2 (BD Bioscience, 551051) and rabbit anti-BCL-x_L (Abcam, ab32370) were used upon crosslinking with Dyna protein G beads (Thermo Fisher, 10004D) using 20 mM dimethyl pimelimidate (Sigma).