Figure 4.

Nephron-specific Jab1 deletion activates the WNK-SPAK/OSR1 pathway. (A, C, and D) Western blot of whole kidney lysates from control and KS-Jab1^−/− mice. (A) Left, immunoblotting was performed with antibodies against WNK4, phosphorylated WNK4 at serine 1196 (pWNK4^S1196), and WNK1. WNK4, pWNK4^S1196, and WNK1 protein abundance was higher in KS-Jab1^−/− mice compared with controls. Right, quantitative analysis of total WNK4 protein abundance and the ratio of pWNK4^S1196 to total WNK4 (pWNK4/tWNK4). Total WNK4 abundance and pWNK4/tWNK4 was higher in KS-Jab1^−/− mice compared with controls. Data represent individual values as well as mean±SEM relative to controls. Statistical differences were examined using two-tailed unpaired t test. (B and E) Immunofluorescence staining of kidney cortex sections from control and KS-Jab1^−/− mice. (B) Immunofluorescence staining was performed with antibodies against WNK4. There was low abundance of WNK4 staining in control mice relative to KS-Jab1^−/− mice. Staining of KS-Jab1^−/− mice kidney sections showed WNK4 protein translocated into puncta. (C) Immunoblotting was performed with antibodies against SPAK and OSR1. Protein abundance of full-length SPAK was higher, whereas SPAK2 and KS-SPAK were lower in KS-Jab1^−/− mice compared with controls. (D) Immunoblotting was performed with antibodies against pSPAK/pOSR1 at serine 373 for SPAK and serine 325 for OSR1. Phosphorylation levels were higher in KS-Jab1^−/− mice compared with controls. (E) Immunofluorescence staining was performed with antibodies against SPAK and OSR1. There was low abundance of SPAK (top) and OSR1 (bottom) staining in control mice relative to KS-Jab1^−/− mice. Staining of KS-Jab1^−/− mice kidney sections showed SPAK/OSR1 protein translocated into puncta.