Figure 5. Efhd1 KO sensory neurons display AMPK and Ulk activation along with increased autophagic flux.

(A) Immunoblot analysis of AMPK and Ulk activation in E13.5. Dorsal root ganglion (DRG) explants were grown on the filter cell culture system for 48 h. AMPK (Thr172), ACC (Ser79), and Ulk (Ser555) phosphorylation status were analyzed. (B) AMPK (Thr172) and Ulk (Ser55) phosphorylation status were analyzed by immunoblot of in vivo (directly extracted) E17.5 DRGs. (C, D, E, F, G) The ratio of phosphorylated to total levels of protein normalized to tubulin was quantified using ImageJ levels in E13.5 (C, D, E) and E17.5 (F, G) DRGs was quantified using ImageJ. (C, D, E) AMPK: soma *P = 0.0168, axons **P = 0.0022, (D) ACC: soma **P = 0.0069, axons *P = 0.0131, (E) Ulk: t test soma *P = 0.0287, axons *P = 0.0185. Graphs show means ± SEM based on three independent experiments (N = 3), unpaired t test was used for all. (F, G) AMPK: t test **P = 0.0038 (G) t test ***P = 0.0005. All graphs show means ± SEM based on N = 4 (p-AMPK) and N = 3 (p-Ulk) independent experiments, unpaired t test was used for both. (H) Immunoblot of LC3I and LC3II levels of WT and Efhd1 KO DRGs treated with bafilomycin (100 nM) for 4 h. (I) The ratio of LC3-II to tubulin was quantified with ImageJ. Graphs show means ± SEM based on three independent experiments (N = 3) (unpaired t test **P = 0.006).

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