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. 2020 Oct 26;130(12):6379–6394. doi: 10.1172/JCI94171

Figure 4. KLHL3 interaction with WNK1 isoforms in HEK293T cells: KLHL3 ubiquitinates KS-WNK1 and significantly reduces its protein levels.

Figure 4

(A) Flp-In T-Rex 293 cells stably and inducibly expressing (His)6-protein C-Flag-hKLHL3 were transfected with myc-tagged L-WNK1 (WT or D635N mutant) or KS-WNK1 (WT or D228N mutant), as indicated. At 34 hours after transfection, cells were induced with tetracycline. Fourteen hours later (48 hours after transfection), cells were harvested and lysed in denaturing conditions. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of 3 independent experiments. (B) Flp-In T-Rex 293 cells stably and inducibly expressing (His)6-protein C-Flag-hKLHL3 were transfected with ubiquitin-HA and myc-tagged L-WNK1, L-WNK1 D635N, KS-WNK1 or KS-WNK1 D228N, as indicated. At 34 hours after transfection, cells were induced with tetracycline. Fourteen hours later (48 hours after transfection), cells were harvested and lysed in denaturing conditions. Upper panel: Myc-tagged WNK1 isoforms were immunoprecipitated with anti-myc antibody (9B11, Cell Signaling Technology); immunoprecipitates were analyzed by immunoblotting with anti-HA antibody (3724S; Cell Signaling Technology). Nitrocellulose membranes were stripped and reblotted with anti-myc antibody. Immunoblot of cell lysates is represented in D (input). Data shown are representative of 3 independent experiments. (C) Cells were transfected with myc-tagged L-WNK1 (WT or D635N mutant) and KS-WNK1 (WT or D228N mutant), as indicated and in conditions similar to those in A. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Densitometric analysis was performed using FUJI FILM Multi-Gauge software. Results are shown as mean ± SEM. *P < 0.05 compared with control, unpaired Student’s t test. n = 3. (D) Flp-In T-Rex 293 cells stably and inducibly expressing (His)6-protein C-Flag-hKLHL3 were transfected with myc-tagged L-WNK1, L-WNK1 D635N, KS-WNK1, or KS-WNK1 D228N, as indicated. At 43 hours after transfection, cells were induced with tetracycline and simultaneously treated with MG132 for 5 hours. At 48 hours after transfection, cells were harvested and lysed in native conditions. Left panel: cell lysates were immunoprecipitated with anti-myc antibody, and immunoprecipitates were analyzed by immunoblotting with anti-protein C and anti-myc antibodies. Right panel: cell lysates (input) were subjected to immunoblot analysis with anti-myc and anti–protein C antibodies (HPC4, Roche) to check for even expression of KLHL3. Data shown are representative of 3 independent experiments.