(A-B) Changes in endogenous DLK abundance in response to treatment with forskolin (30 µM) (A) or the PKA inhibitor H-89 (5 µM) (B) for 6 hr in cultured rat embryonic cortical neurons. Quantification shows relative DLK/Tubulin levels in western blots. (C) Alignment of activation loop sequences in different species. (D) The anti-pDLKS302 antibodies recognize transfected Flag-DLKWT, but not activation loop mutation Flag-DLKS301A,S305A. Both proteins were transiently expressed in HEK293 cells. Western blots were probed with anti-pDLKS302 antibody, anti-Flag antibody to detect the total DLK expression levels, and anti-Tubulin (which remains similar in all manipulations) for normalization. (E) PKACA stimulates phosphorylation of DLK S302 in HEK293 cells. HEK293 cells were co-transfected with Flag-tagged DLKWT and an empty control plasmid or PKACA. Cell lysates were probed with anti-DLK antibody, anti-pDLKS302 antibody, anti-PKA C antibody and anti-tubulin antibody. (F) PKACA stimulates an increase in DLK molecular weight. Flag-DLK protein was immunoprecipitated from HEK293 cells co-transfected with DLK and either Flag-tagged DLKWT and an empty control plasmid or PKACA. The immunoprecipitated Flag-DLK was then incubated with either glycerol (control) or lambda protein phosphatase (λPP). PKACA induced a upward shift in DLK molecular weight, which was lost upon phosphatase treatment. (G) The activation loop is required for axonal regeneration in Drosophila neurons. Single axons in Drosophila third instar larva are labeled by mCD8RFP using eve-Gal4 driver. 24 hr after injury, these neurons in animals heterozygyous for wnd (wnd3/+) show robust axonal sprouting. However, sprouting fails to occur in wnd3/wnd3 animals. Expression of UAS-Wnd (WT) can restore axonal regeneration in wnd mutant background (UAS-Wnd, wnd3; wnd3,eve-Gal4, UAS-mCD8RFP). However, expression of activation loop mutant UAS-WndS301A,S305A failed to rescue the sprouting defect in wnd mutant animals (UAS-WndS301A,S305A, wnd3; wnd3, eve-Gal4,m12-mCD8RFP). Quantification of the volume of axonal membrane within 100 µm of the distal ending of the proximal stump. n> 50 axons for each genotype. Data are presented as mean ± SEM for 3 independent experiments; ***p<0.001; scale bar, 100 µm.