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. 2017 Jan 17;114(5):E879–E886. doi: 10.1073/pnas.1620315114

Fig. 3.

Fig. 3.

Characterization of WNK4 phosphorylation dynamics using phosphosite-specific antibodies. (A) COS-7 cells transfected with WNK4-HA and AT1 were stimulated with the indicated drugs for 30 min. Lysates were immunoblotted with the indicated antibodies. pS47, pS64, pS1169, pS1180, and pS1196 are WNK4 phosphosite-specific antibodies. (B) Results of quantitation of blots in A (n ≥ 4, in at least three independent experiments). Band intensity values obtained with ImageJ were normalized, establishing vehicle, BIM, or H89 groups as 100%. (C) Immunopurified WNK4-HA was incubated with recombinant αPKC and PKA in the presence of all of the components necessary for kinase activity (30 min). Proteins were blotted with the indicated antibodies. See also Fig. S2.