Fig. 6. Acute Rab35 depletion results in mTORC1 hyperactivation in vivo.
a, b p-S6 and p-S6K levels in Schwann cell/DRG neuron co-culture explants transduced with scrambled (scr) or Rab35-targeting shRNA lentiviruses. a Representative immunoblot of lysates from shRNA-transduced co-cultures. b Quantification of representative data in a. Scr were set to 1. One sample two-tailed Student’s t-test with a theoretical mean of 1: p-S6K/Vinculin: **p = 0.0092, t = 10.35, df = 2, n = 3 DRG pools of at least 10 explants each; p-S6/Vinculin: *p = 0.0286, t = 3.35, df = 4, n = 5 DRG pools of at least 10 explants each. c–e Immunoblot analysis of sciatic nerves from P30 Rab35 control (Rab35flox/flox) and cKOSC (Rab35flox/flox P0-Cre) mice. c Representative immunoblot for p-S6, total S6, p-S6K, and total S6K. Tubulin, Vinculin as loading controls. d Quantification of representative data in c. Controls were set to 1. One sample two-tailed Student’s t-test with a theoretical mean of 1: p-S6/S6: *p = 0.0253, t = 1.455, df = 3, n = 4 pools of nerves per genotype and independent experiments; p-S6K/S6K: p = 0.0706, t = 2.291, df = 5, n = 6 pools of nerves per genotype from four independent experiments. e Representative immunoblot for p-AKT, total AKT, S44/42-phosphorylated ERK (p-ERK), and total ERK in sciatic nerve lysates. Vinculin was analyzed as a loading control. f, g Rab35 protein levels are decreased in sciatic nerve lysates from Mtmr2 KO mice. Sciatic nerves from Mtmr2 WT (Mtmr2+/+) and KO (Mtmr2−/−) mice at P30 were lysed and analyzed by immunoblotting, f Representative immunoblot for Rab35, and tubulin as a loading control. g Quantification of representative data shown in f. Data are from n = 4 pools of nerves per genotype from four animals per genotype; One sample two-tailed Student’s t-test with a theoretical mean of 1, p = 0.0789, t = 2.622, df = 3. Data represent mean ± SEM. See Source Data file for numerical source data and unprocessed blots.