Fig 3. Wnk signals through Fray to regulate glial potassium flux.

(A) Representative Western blots stained for phosphorylated rat Ste20-related proline/alanine-rich kinase (p-SPAK), total rat Ste20-related proline/alanine-rich kinase (t-SPAK), and β-actin. The pan glial driver Repo-Gal4 was used to express kinase-dead rat SPAK (RnSPAK^D219A) as a substrate for endogenous Drosophila Wnk. Western blots were first probed for p-SPAK, stripped, and re-probed for t-SPAK. (B) Quantification of the mean (±SEM) ratio of pSPAK/total SPAK for genotypes in (A). Phosphorylation of SPAK decreased with Wnk knockdown (Repo>RnSPAK^D219A,Wnk RNAi) and increased when Wnk was overexpressed (Repo>RnSPAK^D219A, Wnk^WT). Data points represent biological replicates of separate lysate preps. Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons test. (C) Representative images of larval peripheral nerves stained for membrane marker HRP. Repo-Gal4, a pan glial driver, was used to express a control transgene, UAS-LexA RNAi (Repo>), UAS-Wnk RNAi (Repo>Wnk RNAi), UAS-Fray RNAi (Repo>Fray RNAi), and co-expression of Wnk RNAi and constitutively active Fray (Repo> Wnk RNAi, Fray^T206E). Loss of Wnk and Fray in glia causes nerve swellings (arrow). Swellings are suppressed by expressing constitutively active Fray. Scale bars 20μM. (D) Quantification of nerve swellings for genotypes in (C). n≥ 20. Statistical significance was determined by one Way ANOVA with Dunnett’s multiple comparisons; ****, p<0.0001.