Fig. 5. Acute Rab35 depletion causes mTORC1 hyperactivation.

Astrocytes from tamoxifen-inducible Rab35 cKO (Rab35^flox/flox; CAG-Cre^ER) or non-Cre^ER-expressing control mice (Rab35^flox/flox, WT) were treated with tamoxifen and serum-fed (+FBS) or starved (−FBS) as indicated. a Representative immunoblot for p-S6K, total S6K, and ß-actin. b p-S6K/total S6K ratio in WT (set to 1) and cKO astrocytes. n = 9 independent experiments. One sample two-tailed Student’s t-test with a theoretical mean of 1; +FBS: *p = 0.0124, t = 3.211, df = 8; −FBS: **p = 0.0093, t = 3.404, df = 8. c, d Representative immunoblot of WT and Rab35 cKO astrocyte lysates probed for p-S6K, total S6K, p-AKT, total AKT, Rab35, and ß-actin (c). d p-AKT/AKT ratio in WT (set to 1) and cKO astrocytes. n = 12 independent experiments. One sample two-tailed Student’s t-test with a theoretical mean of 1; **p = 0.0022, t = 3.965, df = 11. e Area of WT and cKO astrocytes shown in Supplementary Fig. 5g. n = 10 independent experiments with WT set to 100. One sample two-tailed Student’s t-test with a theoretical mean of 100; ***p = 0.0004, t = 5.411, df = 9. f p-S6K/total S6K ratio in Rab35 WT (set to 1) and cKO astrocytes transduced with lentiviruses encoding scrambled (scr) or anti-MTMR2 shRNA from n = 6 independent experiments. See Supplementary Fig. 5p. One sample two-tailed Student’s t-test with a theoretical mean of 1, for all conditions normalized to control (WT + scr), followed by Holm’s Multiple Comparison Test to correct for multiple testing; cKO + scr: *p = 0.0209, t = 3.32454, df = 5; WT + shMTMR2: **p = 0.0047, t = 6.21728, df = 5; cKO + shMTMR2: *p = 0.0205, t = 4.00827, df = 5. g MTMR2 levels in lysates from Rab35 WT and cKO astrocytes. See representative data in Supplementary Fig. 5s. n = 8 independent experiments. One sample two-tailed Student’s t-test with a theoretical mean of 100, **p = 0.0013, t = 5.17, df = 7. All data represent mean ± SEM. See Source Data file for numerical source data and unprocessed blots.