The JNK pathway and microtubule stability are downstream of Sarm1 and Sdc2. (A) Sarm1 overexpression increases the acetylation levels of tubulin. COS-1 cells were transfected with vector control or Sarm1 and were immunoblotted with acetyl-tubulin and α-tubulin antibodies. The levels of tubulin acetylation were normalized to total α-tubulin. (B) Sarm1 knockdown reduces tubulin acetylation in mouse neuroblastoma Neuro-2A cells. 2 d after transfection, the acetylation levels of tubulin were determined by immunoblotting. (C) A JNK kinase dead mutant (JNK^KR) and an MKK4 dominant-negative mutant (MKK4^DN) suppress the effect of Sarm1 overexpression in dendritic arborization. (D) Sarm1-expressing neurons treated with JNK inhibitor SP600125 (SP) have shorter dendrites compared with Sarm1-expressing cells. In C and D, transfection was performed at 9 DIV and cells were harvested for immunostaining at 12 DIV. (E) Co-expression of JNK^KR abolishes Sdc2–induced dendrite outgrowth. At 2 DIV, cultured hippocampal neurons were transfected with the indicated constructs, then fixed for immunostaining at 5 DIV. Three independent experiments were performed. Equal numbers of transfected neurons were analyzed for each experiment. The data represent mean values ± SEM (error bars). *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Bars, 30 µm.