Figure 7

Dephosphorylation of Bcl-2 at Thr69 by CDC25A is required in inhibition of apoptosis by NPAS2. (a) Western blot analyses for NPAS2, Bax, Bak, Bcl-2 and Bcl-xl in HLE and HLF cells with treatment as indicated. (b) qRT-PCR analyses for mRNA expression levels of Bcl-2 in both HLE and HLF cells with treatment as indicated. (c) Western blot analyses for NPAS2, CDC25A, phosphorylated Bcl-2 at T69 (p-Bcl-2) and Bcl-2 in HLE and HLF cells treated as indicated. (d) Western blot analyses for CDC25A, p-Bcl-2 (T69) and Bcl-2 in HLF cells transiently transfected with a control vector, or CDC25A, or phosphatase-inactive CDC25A (C431S). (e and f) Co-IP assay using control IgG and anti-CDC25A or anti-Bcl-2 antibody was carried out using extracts prepared from HLF cells. The presence of CDC25A or Bcl-2 in these IPs was evaluated by immunoblotting (WB). Experiments were repeated twice with independent cell extracts and representative data were presented. Data shown are the mean±S.E.M. from three independent experiments. *P<0.05; **P<0.01