Cypa bml sa296

Preparation of total protein lysates and western blot analysis was done as previously described [46 (link)]. The following antibodies were used: Mcl-1 (S-19), Bcl-2 (N-19), MDR (H-241), AIF (E-1), actin (C-11), and horseradish peroxidase-conjugated secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA); Bcl-xL (#610211), cytochrome c (7H8.2C12), Smac (#612245) from BD Biosciences (San Jose, CA); cleaved PARP (9541), CoxIV (#4844) from Cell Signaling Technology (Danvers, MA); and CypA (BML-SA296) from Enzo Life Sciences. After immunodetection, our preference for loading controls was for staining of total proteins transferred to the membrane with Coomassie blue because drug treatments often affect the levels of typical housekeeping proteins such as actin or tubulin.

The cell lysate was resolved by SDS polyacrylamide gel electrophoreses. Proteins were transferred onto nitrocellulose and blocked subsequently with 5% milk in Tris-buffered saline/0.1% Tween20 (TBS/T) for 1 h. Next, blots were incubated overnight at 4 °C with appropriate antibodies: rabbit polyclonal acK antibody (#9441, Cell Signaling, Frankfurt am Main, Germany); rabbit polyclonal CyPA (BML-SA296, EnzoLife Science, Lörrach, Germany); p-P38 (#9211, Cell Signaling, Frankfurt am Main, Germany); β-Actin (#1616, Santa Cruz, Dallas, TX, USA); GPαIIb (kind gift of B. Nieswandt, University of Würzburg, Germany).
After washing 3 times with TBS/T, membranes were probed with secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody (Biorad, Feldkirchen, Germany), and rabbit HRP protein A (PA1-26853, Thermo Fischer Scientific, Waltham, MA, USA), and visualized using enhanced chemiluminescence reagent (GE Healthcare, München, Germany). Chemiluminescence images were taken using the Amersham Imager 600 (GE Healthcare, München, Germany). Uncropped Western Blot images are provided as Supplementary Data.