Fig. 3.

R1185C mutation suppressed CaM binding ability of WNK4 COOH-terminal region. A: R1185C mutation greatly inhibited the binding of GST-WNK4 1163–1212 to CaM-agarose beads (left). Coomassie Blue staining indicated equal loading of proteins between wild-type (WT) and R1185C mutant of GST-WNK4 1163–1212 (right). B: CaM binding ability of GST-WNK4 1163–1243 was significantly suppressed by the R1185C mutation as demonstrated by far-Western overlay with [³⁵S]-labeled CaM probe. Densitometric analyses of far-Western results (n = 3) are shown at right. *P < 0.05 vs. WT group. C: [Ca²⁺] dependence of [³⁵S]-CaM binding to GST-WNK4 1163–1243 WT and R1185C mutant demonstrated by far-Western blot analyses. Bottom: means ± SE of band intensities from 5 experiments.