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. 2011 Aug 1;22(15):2664–2679. doi: 10.1091/mbc.E11-04-0374

FIGURE 5.

FIGURE 5.

Effect of agonist activation of the hIP on the interaction and phosphorylation of PDZK1. (A) HEK.hIP cells, transiently transfected with pCMVTag2C:PDZK1, were stimulated with cicaprost (1 μM; 0–240 min). HA-tagged hIPs were immunoprecipitated with anti-HA 101R antibody; immunoprecipitates (IP) were resolved by SDS–PAGE and immunoblotted (IB), as indicated. The bar charts show mean relative levels of the PDZK1 associated with the anti-HA.hIP 101R immunoprecipitates as a function of cicaprost stimulation (relative protein, % ± SEM, n = 3) where levels in the absence of agonist are expressed as 100%. The asterisks indicate that cicaprost stimulation resulted in significant reductions in levels of PDZK1 associated with the hIP immune-complexes where * and ** indicate p < 0.05 and p < 0.01, respectively, for post hoc Dunnett's multiple comparison t-test analysis. (B–E) HEK.hIP cells, transiently transfected with either pCMVTag2C:PDZK1 (B–D) or pCMVTag2C:PDZKS505A (E), were preincubated with vehicle (B, E), RO1138452 (10 μM; 10 min) (C), or H-89 (10 μM; 10 min) (D) prior to stimulation with cicaprost (1 μM; 0–240 min). Cells were then subject to immunoprecipitation with anti-FLAG antibody to immunoprecipitate PDZK1. Immunoprecipitates (IP) were resolved by SDS–PAGE and immunoblotted (IB), as indicated.