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. 2018 May 30;315(3):F734–F745. doi: 10.1152/ajprenal.00145.2018

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Fig. 7. — Kidney-specific with no lysine kinase 1 (KS-WNK1) promotes WNK4 S335 phosphorylation through heterodimer formation. A: representative Western blot of the total lysate and KS-WNK1 lacking exon 11 (KS-WNK1-Δ11) immunoprecipitation (IP) from oocytes injected with WNK4 cRNA alone or together with KS-WNK1-Δ11 cRNA or KS-WNK1-Δ11 HQ/AA, as stated. IP was performed using the c-Myc tag antibody, which exclusively recognizes KS-WNK1-Δ11. Left: blot from total lysate shows total WNK4 and pWNK4 expression, as stated. Lanes were loaded as follows: lane 1: H₂0-injected oocytes; lane 2: WNK4-injected oocytes; lane 3: WNK4-injected oocytes exposed to low-chloride hypotonic stress (LCHS) conditions; lane 4: WNK4 + KS-WNK1-Δ11-injected oocytes; lane 5: WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Right: blot from the IP assay. IP was performed against the c-Myc tag, which recognizes KS-WNK1-Δ11 and revealed against WNK4 or pWNK4, as stated. Lanes were loaded as follows: lane 1: IP from WNK4 + KS-WNK1-Δ11-injected oocytes; lane 2: flowthrough of WNK4 + KS-WNK1-Δ11-injected oocytes; lane 3: IP from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes (the absence of WNK4 in the IP confirms the specificity of the c-Myc IP); lane 4: flowthrough from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Identical results were observed in 3 independent experiments. B: representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 L322F cRNA or WNK4 S335A alone or together with KS-WNK1-Δ11 cRNA, as stated. Left: blot from total lysate shows total WNK4 and pWNK4, as stated. Right: blot from the IP assay. Lanes 1 and 3 were loaded with proteins from the IP fraction, whereas lanes 2 and 4 were loaded with proteins from the flowthrough, as stated. The blot shows pWNK4 and WNK4 expression, as stated. Identical results were observed in 3 different experiments. C: representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 S335A cRNA together with KS-WNK1-Δ11 or KS-WNK1-Δ11-Δ4a cRNA, as stated. Blot from the IP assay is shown. Lanes 1, 3, and 5 were loaded with proteins from the flowthrough, whereas lanes 2, 4, and 6 were loaded with proteins from the IP fraction, as stated. The blot shows total WNK4 and pWNK4 expression, as stated. Identical results were obtained from 3 independent experiments.