Figure 5
Akt-mediated phosphorylation of CDK2 was enhanced in CSN5-depleted cells.
(A), (B) CSN5-floxed p53-null MEFs containing CRE-ER (as in Fig. 3) were cultured with and without 4OHT and passaged in a normal medium for 4 days. Cells were incubated in low serum for 48 hours and stimulated with serum. Lysates isolated from cells at indicated times after serum stimulation were analyzed by Western blotting using antibodies against CSN5, MEK1, MEK2, phosphorylated MEK1/2, Erk1, Erk2, phosphorylated Erk1/2, Akt, phosphorylated Akt (Ser473 and Ser 308), phosphorylated Akt substrate and γ-tubulin. (C) CSN5-floxed p53-null MEFs containing CRE-ER were cultured with and without 4OHT and then in a normal medium for 6 days. Cell lysate was isolated and analyzed by Western blotting using antibodies against phosphorylated Akt substrate and γ-tubulin. (D) Cell lysate as in C was immunoprecipitated with NRS and rabbit antibodies raised against CDK2. The immune complex was analyzed by Western blotting using antibodies against CDK2 and phosphorylated Akt substrate. (E–G) CSN5-floxed p53-null MEFs containing CRE-ER were cultured with and without 4OHT and then in a normal medium for 6 days in the presence and absence of wortmannin. Cell lysate was isolated and analyzed by Western blotting using antibodies against Akt, phosphorylated Akt (Ser473 and Ser 308) and γ-tubulin (E). Cells were fixed and stained with an antibody to CDK2. DNA was visualized by staining with Hoechst 33342 (Original magnification; x200)(F). Cells with cytoplasmic staining of CDK2 were enumerated (G).
