Fig. 3

Bok mutants show that PKA-mediated Bok phosphorylation is limited to Ser-8. HEK-3KO and HeLa BKO cells were transfected to express mouse (m) or human (h) Bok^WT, Bok^S8A, or Bok^S8E (0.25 µg cDNAs). (A and B) Lysates were probed in the immunoblots as indicated, with p97 serving as a loading control. Histograms show immunoreactivity seen with Bok^A, Bok^B, and Bok^C, normalized to mBok^WT or hBok^WT (mean ± SEM, n = 4, *, **, and *** designates p < 0.05, p < 0.005, and p < 0.0005, respectively, ns = not significant, p > 0.05). (C and D) HEK-3KO and HeLa BKO cells expressing hBok^WT, hBok^S8A, or hBok^S8E were treated with 1 µM PGE1 or 10 µM isoproterenol, respectively, for the times indicated. Lysates were probed in the immunoblots as indicated, with Mcl-1 and phospho-proteins serving as controls for the methodology, and p97 serves as a loading control. Histograms show Bok immunoreactivity seen with Bok^A, Bok^B and Bok^C, normalized to 0 min controls (mean ± SEM, n = 3). (E and F) Immunoblots of lysates or immunoprecipitated PKA substrates (RRXpS IP) from HEK-3KO and HeLa BKO cells expressing hBok^WT, hBok^S8A, or hBok^S8E exposed to 100 nM CalA and 20 µM Fsk for 10 min, or 1 µM PGE1 or 10 µM isoproterenol for 2.5 min. Samples were probed in the immunoblots as indicated, with p97 serving as a loading control and IgG light chain (25 kDa) is labeled with an asterisk