S2 cells were maintained at 25 °C in Schneider’s Drosophila Medium (S9895, Sigma) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (P0781, Sigma). Transfection was performed using calcium phosphate according to the manufacturer’s instructions (Invitrogen). An ub-gal4 plasmid was co-transfected with pUAST expression vectors for all experiments. 48 hrs after transfection, cells were harvested for immunoprecipitation and western blot analysis with standard protocols (described in Molecular Cloning). The primary antibodies used were mouse anti-HA (F7) (1:2500; Santa Cruz); rabbit anti-HA (Y11) (1:2500; Santa Cruz), mouse anti-Myc (9E10) (1:2500; Santa Cruz); mouse anti-Fg (1:2500; Sigma); mouse anti-Ub (P4D1) (1:1000; Santa Cruz); rabbit anti-HIB (1:1000; ABclonal Technology) and mouse anti-Actin (A00702) (1:5000: Genscript). rabbit anti-HIB antibody was generated in rabbit with full length HIB protein as antigen (from ABclonal Technology). After incubation with HRP-coupled secondary antibodies (goat anti-mouse diluted 1:10000, Abmax; goat anti-rabbit diluted 1:10000, Jackson ImmunoResearch), the blots were visualized using a chemiluminescent detection kit (GE healthcare).
Zhou Z., Xu C., Chen P., Liu C., Pang S., Yao X, & Zhang Q. (2015). Stability of HIB-Cul3 E3 ligase adaptor HIB Is Regulated by Self-degradation and Availability of Its Substrates. Scientific Reports, 5, 12709.
+ Open protocol