Anti β actin clone ac 15

Antibodies for MCL-1-BIM co-immunoprecipitation studies were obtained from Thermo Fisher Scientific (Waltham, MA, USA; anti-MCL-1 clone RC13) and Abcam (Cambridge, MA, USA; anti-BIM clone Y36). Secondary anti-rabbit-HRP and anti-mouse-HRP antibodies were purchased from Amersham Biosciences (Piscataway, NJ, USA). All chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies for immunoblots were obtained from Abcam (anti-NOXA clone 114C307), BD Biosciences (San Jose, CA, USA; anti-BCL-X_L clone 4), Sigma (anti-β-actin clone AC-15) and Santa Cruz (La Jolla, CA, USA; anti-MCL-1 clone S-19).

Cells were harvested and washed in ice-cold PBS and suspended in RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma Aldrich). The insoluble debris was removed by centrifugation at 14,000 g for 30 min. Cellular extracts were resolved using 12% SDS-PAGE and transferred to PVDF membranes (Hybond-P, Amersham). Immunoblot analysis was performed using anti-hTS (clone TS106, Millipore, 1:500 dilution), anti-hDHFR (clone 872442, Millipore, 1:1000) and anti-FLAG M2 (clone M2, F1804, Sigma-Aldrich, 1:1000 dilution), anti-β-Actin (clone AC-15, Santa Cruz Biotechnology, 1:1500 dilution). Horseradish peroxidase-conjugated secondary antibody (GE Healthcare UK Limited) was used to detect the bound primary antibody. Immune complexes were visualized by enhanced chemiluminescence (Amersham ECL Prime Western blotting reagent) following the manufacturer’s instructions. Band density was calculated using Image J software or an image analyzer (GS-690 BIORAD).

Western blotting was performed as previously described.6 Briefly 10⁷ Jurkat T cells were lysed in 200 μl Triton X‐100 buffer and 2·5 × 10⁵ to 7·5 × 10⁵ cells were resolved by SDS–PAGE under reducing conditions. Protein bands were detected by the LI‐COR Odyssey Sa system after developing with rabbit anti‐SAP antibody (clone FL‐128 Santa Cruz Biotechnology (Santa Cruz, CA)), mouse anti‐phosphotyrosine (clone PT‐66, Sigma, St Louis, MO), goat anti‐HA (biotinylated, Vector Laboratories) or anti‐β‐actin (clone AC‐15, Santa Cruz Biotechnology) followed by the appropriate secondary antibody or fluorophore‐conjugated streptavidin (IRDye 680LT‐conjugated anti‐rabbit IgG, IRDye 800 CW anti‐mouse IgG, IRDye 680LT‐conjugated anti‐mouse IgG, IRDye 800 CW Streptavidin; all LI‐COR Biosciences, Lincoln, NE). Quantification of protein expression was performed using LI‐COR odyssey sa software version 1.0.