Mouse anti bax

Treatments with 100 μM GroPIns, 35 μM Fludarabine, 3.5 nM Venetoclax or combination treatments were carried out at 37°C in RPMI 7.5% BCS for the indicated times. Control samples were treated with DMSO (Merck Millipore, #102952). Dose-response and time course experiments of CLL B cells treated with GroPIns are shown in Supplementary Figure 2. When required, cells were pretreated at 37°C for 20 min with 50 μM NSC-87887. Cells (5×10⁶ cells/sample) were lysed in 1% (v/v) Triton X-100 in 20 mM Tris-HCl pH 8, 150 mM NaCl, in the presence of a cocktail of protease inhibitors (Calbiochem, #539134) and 0.2 mg/ml Na orthovanadate (Merck, #S6508), resolved by SDS-PAGE and transferred to nitrocellulose (GE Healthcare, #9004-70-0). Immunoblots were carried out using mouse anti-Bax (BD Biosciences, #610982), anti-penta-His (Life Technologies, #P21315) and anti-actin (Millipore, #MAB1501) primary antibodies. Secondary peroxidase-labeled anti-mouse antibodies were from Jackson Immuno-Research (#115-035-146). Labeled antibodies were detected using ECL kit (SuperSignal^® West Pico Chemiluminescent Substrate, Thermo Scientific) and scanned immunoblots were quantified using the ImageJ software.

Western blot analysis was performed as described previously [28 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-Noxa, rat anti-Bmf (Alexis Biochemicals, Grünberg, Germany), mouse anti-Bcl-2, rabbit anti-Bcl-x_L, mouse anti-Bax, rabbit anti-Bak (BD Transduction Laboratories, Heidelberg, Germany), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-Bim, mouse anti-PARP (Cell Signaling, Beverly, MA), acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-Mcl-1 (Stressgene, Victoria, BC), rabbit histone H3 (Abcam, Cambridge, UK). Mouse anti-GAPDH (HyTest, Turku, Finland), mouse α-Tubulin (Calbiochem, Darmstadt, Germany) or mouse β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) were used as secondary antibodies. Enhanced chemiluminescence (Amersham Bioscience, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odyssey Imaging System, LICOR Bioscience, Bad Homburg, Germany) were used for detection. Representative blots of at least two independent experiments are shown.