The involvement of survival signaling pathways in rubella-virus induced apoptosis

Cooray, Samantha; Jin, Li; Best, Jennifer M

Polyclonal anti-PI3K p85, anti-HA Tag, anti-myc Tag, and monoclonal anti-β-tubulin antibodies were from Upstate Biotechnology inc. (UK). Polyclonal anti-caspase-3 antibody was from Sigma (UK). All other primary antibodies were purchased from Cell Signaling Technology (UK). Cells were treated as described above and at indicated times post-infection (p.i.), washed in PBS and harvested in cell lysis buffer [50 mM Tris, 150 mM NaCl, 1% Triton-X-100, 2 mM EDTA, 2 mM EGTA, 100 µM protease inhibitor cocktail, and 100 µM each of phosphatase inhibitor cocktails 1 and 2 (Sigma, UK)]. Protein concentrations were determined using the BioRad assay (BioRad, Hemel Hemstead, UK), and equal protein loading was determined by Coomassie staining (Invitrogen, Paisley, Scotland). Lysates were electrophoresed on 12% Bis-Tris polyacrylamide gels (Invitrogen, UK) and transferred onto Hybond™ ECL nitrocellulose or PVDF membranes (Amersham Biosciences, UK). Membranes were blocked with 5% non-fat dried milk in PBS containing 0.1% Tween-20, and subsequently incubated with primary antibody (1:1000) overnight at 4°C. Specific antibody binding was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (1:2000) (Dako, UK), and immunoreactive bands were visualized using the ECL detection system according the manufacturer's instructions (Amersham Biosciences, UK).