(A, B) HEK293 cells were transfected with 0.5 μg of HA-tagged PKCα, δ, ε, or ζ in (A) and with 20 nMsiRNAs against PKCα, δ, ε, or ζ in (B). (C, D) Time-course stimulation of FGF2. Protein levels (C) and mRNA levels (D) of Sur8 and PKC isotypes were assessed by immunoblot and RT-PCR, respectively. (E) FGF2-dependent interaction of Sur8 with PKCα/δ. Cells were treated with FGF2 for the indicated time and MG132 for 4 hours before WCEs were collected for immunoprecipitation with either IgG control or anti-Sur8 antibody. (F) Cells were transfected withHA-Ub-K48 plasmid and treated with FGF2 for 12 hours and MG132 for 4 hours as indicated. WCEs were immunoprecipitated with anti-PKCα or PKCδ antibody. (G, H) Effects of PKCα/δ OE or KD on FGF2-mediated Sur8 stabilization. Cells were transfected with the indicated plasmids or siRNAs and treated with or without FGF2 for 24 hours at 24 hours post-transfection. WCEs were subjected to immunoblot analysis (A-C, E-H).