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. 2022 Jul 22;119(30):e2203743119. doi: 10.1073/pnas.2203743119

Fig. 2.

Fig. 2.

WNK1 stabilizes the TGF-β–sensitive angiogenic tyrosine kinase AXL. (A) Western blot quantification shows decreased AXL expression upon WNK1 siRNA (control siRNA) depletion or siControl in HUVECs; n = 4. (B) Representative Western blots show AXL expression in cells treated with SW113708 (10 µM) overnight. (C) Corresponding quantification of the Western blots in B shows decreased AXL expression upon SW113708 treatment compared with DMSO control in HUVECs; n = 4. (D) Western blot quantification shows decreased AXL expression upon overnight WNK463 treatment compared with DMSO control in HDMECs; n = 5. (E, Left) Representative Western blot shows full-length and cleaved fragment of AXL upon treatment with DMSO control or the proteasomal inhibitor MG132 (10 µM) with or without WNK463 (1 µM) cotreatment in HUVECs. (Right) Representative Western blots show AXL expression in HUVECs treated with DMSO control or WNK463 (1µM) overnight and co-treatment with or without the SMURF1 inhibitor A01 (2µM) or TGFβ receptor inhibitor (TGFBRi: galunisertib, 1µM). (F) Corresponding quantification of Left panels in E shows increased accumulation of cleaved fragment of AXL upon WNK463+MG132 treatment compared with MG132 alone; n = 3. (G) Quantification of 18S normalized relative mRNA expression in HUVECs shows no change in mRNA expression of AXL treated with WNK463 compared with DMSO control; n = 6. (H) Corresponding quantification of Right panels in E show differences in AXL expression in cells treated with A01+WNK463 compared to A01 alone; n = 3. (I) Quantification of angiogenic factors secreted into conditional medium 4 h after initiation of cord formation in HUVECs treated with DMSO control or WNK463 (1 µM); n = 3. (J) Model figure shows the collaboration among WNK1 and TGF-β signaling in regulating AXL. Graphics were created with BioRender.com. Data are represented as mean ± SE analyzed by unpaired two-tailed Student’s t test or one-way ANOVA. *P < 0.05, **P < 0.005, ***P < 0.0005.