Fig. 8
From: Phosphorylation of Bok at Ser-8 blocks its ability to suppress IP3R-mediated calcium mobilization
BokS8E or phosphorylation of BokWT at Ser-8 weakens the Bok-IP3R1 interaction. (A) HEK-3KO cells were transfected to express IP3R1HAWT (2 µg cDNA) and either BokWT, BokS8A, BokS8E, or vector (0.25 µg cDNAs). Immunoblots of lysates or IP3R1HA IPs were probed as indicated, with p97 serving as a loading control. Histogram shows co-IP immunoreactivity of the Bok constructs, normalized to BokWT (mean ± SEM, n = 4, * designates p < 0.05, ns = not significant, p > 0.05). (B) Immunoblots of lysates or IP3R1 IPs from HEK-IP3R1 cells stably expressing BokWT, BokS8A, or BokS8E, with vector as a control, were probed as indicated, with p97 serving as a loading control. Histogram shows co-IP immunoreactivity of the Bok constructs, normalized to BokWT (mean ± SEM, n = 7, ** designates p < 0.005, ns = not significant, p > 0.05). (C) HEK-IP3R1 cells stably expressing BokWT were exposed to 1 µM PGE1 for 2.5 or 10 min. Immunoblots of lysates or IP3R1 IPs were probed as indicated, with RRXpS serving as a control for the methodology and p97 as a loading control. Histogram shows co-IP immunoreactivity of Bok, normalized to t = 0 (mean ± SEM, n = 8 for 2.5 min PGE1 and n = 5 for 10 min PGE1, * and ** designates p < 0.05 and p < 0.005, respectively). (D) IP3R1 IPs from HEK-IP3R1 cells stably expressing BokWT were incubated without or with the catalytic subunit of PKA. Immunoblots were probed as indicated, with RRXpS showing that Bok is phosphorylated. Histogram shows co-IP immunoreactivity of Bok, normalized to control (mean ± SEM, n = 3, * designates p < 0.05)