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. 2011 Nov;179(5):2254–2265. doi: 10.1016/j.ajpath.2011.07.046

Figure 3.

Figure 3

Interaction between PC and ezrin is inhibited in PAN-nephrotic glomeruli and in a mutant mimicking phosphorylation of the PKC phosphorylation site (Ser415) of PC. A: GST-N′-Ezrin binds His-PCT directly when the latter is either sham-treated (lane 3) or treated with PKA (lane 9), but binding is markedly reduced when His-PCT is phosphorylated with PKC (lane 6). In vitro binding assays were performed using sham-treated or in vitro phosphorylated 6-His-tagged PC tail (6-His-PCT) with GST-N′-Ezrin immobilized on glutathione S-sepharose beads. Bound proteins were analyzed by immunoblotting with His antibody (IB). Top panel shows an immunoblot, and bottom panel shows Ponceau S staining for the same blot. B: Interaction between endogenous PC and ezrin is decreased in PAN-nephrotic glomeruli. Pull-down assays were performed on glomerular lysates (normal versus 10 days PAN) with GST or GST-N′-Ezrin immobilized on glutathione S-sepharose beads. Bound proteins were analyzed by immunoblotting with PC antibody (0601). GST-N′-Ezrin bound strongly to PC from normal glomerular lysates (lane 3) but weakly to PC from PAN-nephrotic glomeruli (lane 6). Top panel shows an Immunoblot (WB), and the bottom panel shows Ponceau S staining for the same blot. C:In vitro binding assay between 6-His-tagged PC proteins and GST-N′ezrin. 6-His-PCT (aa 411 to 485); ΔN12, a juxtamembrane N terminal truncation mutant (Δ411–422); S415E, a phosphomimetic PC mutant; and S415A, a phospho-deficient PC mutant, were incubated with GST-N′-ezrin, and bound proteins were detected by immunoblotting with anti-PC 0601. PCT, PC S415E and PC S415A, but not PCT-ΔN12 bind to N′-ezrin. D: Densitometric analysis of data in C showing that binding of PC S415A or PC S415E to ezrin is weaker than that of PCT. E:In vitro binding assay between GST-PC tail mutants and in vitro-translated, 35S-labeled N-terminal ezrin. GST-PC and PC S415D and S415E (phosphomimetic mutants of PC) and S415A (a phospho-deficient mutant), were incubated with 35S-labeled, N-terminal ezrin in binding buffer and processed for autoradiography. F: Densitometric analysis of data in E showing that binding of the S415D and S415E phosphomimetic mutants is weaker than that of PCT or the phospho-deficient S415A mutant.