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

Fig. S2.

Fig. S2.

Analysis of WNK4 phosphorylation dynamics in COS-7 cells using phosphosite-specific antibodies. (A) Demonstration of specificity of WNK4 phosphosite-specific antibodies. COS-7 cells were transfected with empty vector, wild-type WNK4, or the indicated mutant forms of WNK4. At 48 h posttransfection, cells were stimulated with TPA for 30 min before lysis and extracts were blotted with the indicated antibodies. The results demonstrate that the antibodies mainly detect the expected phosphorylated epitope. (B) Characterization of the dynamics of WNK4 phosphorylation in HEK293T cells using phosphospecific antibodies. HEK293T cells were transfected with WNK4-HA and 48 h posttransfection they were stimulated with the indicated drugs for 30 min and then lysed. For the AngII experiment, cells were serum-depleted overnight before stimulation. Extracts were immunoblotted with the indicated antibodies. pS47, pS64, pS1169, pS1180, and pS1196 are WNK4 phosphosite-specific antibodies. (C) Results of quantitation of blots in B (n = 3 or higher). Band intensity values obtained were normalized, establishing the vehicle, BIM, or H89 groups as 100%. Data are expressed as means.