Western Blot Analysis of BIM and β-Actin

Cell extracts for western blot analysis were prepared in ONYX lysis buffer (20 mM Tris-HCl pH 7.4, 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 10% glycerol). Antibodies used include: rat anti-BIM (clones 3C5 and CF7, ENZO Life Sciences,^{29 (link)} Waterloo, NSW, Australia), mouse anti-β-actin (Sigma AC-40, Rowville, VIC, Australia). Horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rat IgG antibodies (both from Southern Biotech, Birmingham, AL, USA) served as secondary reagents and the enhanced chemoluminescence (ECL; GE Healthcare, Rydalmere, NSW, Australia) system was used for detection. WBC analysis was performed with an ADVIA blood analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA).