Fig 1: Kinase dead WNK4 D321A or chloride insensitive WNK4 L319F abolish kAE1 effect on tight junctions.A, Immunoblot using mouse anti-HA antibody to detect both WNK4 WT or mutants and kAE1 protein in mIMCD3 cells inducibly expressing kAE1 and transiently transfected with cDNA encoding WNK4 WT, WNK4 D321A or WNK4 L319F. B, Percentage of transepithelial electrical resistance relative to mIMCD3 cells without Dox measured in cells expressing kAE1 (+ or – Dox) and transiently transfected with cDNA encoding WNK4 WT, WNK4 D321A or WNK4 L319F. C, PNa/PCl ratio, PNa and PCl measured for IMCD cells transfected with WNK4 WT. D, PNa/PCl ratio, PNa and PCl measured for IMCD cells transfected with WNK4 D321A. E, PNa/PCl ratio, PNa and PCl measured for IMCD cells transfected with WNK4 L319F. Error bars correspond to means ± SEM, n = 4–6. *P < 0.05, **P < 0.01, not significant (n.s.) using two-way ANOVA and a Fisher's LSD test. Uncropped blots are provided in Supplementary Figures.
Fig 2: mIMCD3 cells express endogenous WNK4, and kAE1 WT but not E681Q mutant protein expression increases WNK4 phosphorylation.A, C, Immunoblots using rabbit anti-total WNK4, rabbit anti-phospho (S1196) WNK4 or mouse anti β-Actin antibodies to detect total, phospho- WNK4 or β-Actin, in kAE1 WT- or E681Q-expressing mIMCD3 cells, respectively, in the presence or absence of kAE1 WT proteins. B, D, quantification of total and phospho-WNK4 normalized to β-actin. Error bars correspond to means ± SEM, n = 4. Not significant (n.s.), *P < 0.05, **P < 0.01 using a Student's t-test or a Welch t-test (phospho-WNK4 in WT cells). Normality of the data was verified using a Shapiro-Wilk test. Note that additional bands may appear on the actin blot, corresponding to detection of the proteins with antibodies incubated prior to the anti-actin antibody. Uncropped blots are provided in Supplementary Figures.
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