Supplementary Figure 9: Unprocessed images of all gels and blots.

Lyso-IP (upper, Fig. 1e). OSBP depletion (lower left, Fig. 2d) and OSW-1 (lower right, Fig. 2e) inhibits sterol-induced mTORC1 signaling. OSBP acts upstream of the Rag GTPases (upper, Fig. 3d), GATOR1 (lower left, Fig. 3e) and KICSTOR (lower right, Fig. 3f). OSBP activates mTORC1 via lysosomal contact (upper, Fig. 4d) and lipid transport (lower, Fig. 4e). Excess PI4P does not inhibit mTORC1 in OSBP-depleted cells (Fig. 5d). VAPA/B (upper, Fig. 6c), but not STARD3 (lower left, Fig. 6d) and ORP1L (lower right, Fig. 6e), regulate sterol-dependent mTORC1 signaling. NPC1 regulates sterol-induced OSBP-VAP binding (left, Fig. 7b). OSBP depletion abolishes hyperactive mTORC1 in NPC1-null cells (right, Fig. 7c). OSBP inhibition by OSW-1 (left, Fig. 8b) and shRNA (right, Fig. 8c) restores autophagy in NPC1 fibroblasts. GFP-OSBP expression in HEK-293A cells (left, Supplementary Fig. 3a). Validation of VAPA/B knockdown (right, Supplementary Fig. 3d). Western blots of Lyso-IP (left, Supplementary Fig. 4a). Validation of OSBP knockdown (right, Supplementary Fig. 4b). Basal state (left, Supplementary Fig. 6f) and amino acid-induced (right, Supplementary Fig. 6g) mTORC1 signaling in OSBP-depleted cells. Truncation (left, Supplementary Fig. 7b) and mutation (right, Supplementary Fig. 7c) analysis of OSBP on mTORC1 activation by sterol. OSW-1 activates autophagy in NPC1 fibroblasts (left, Supplementary Fig. 8c; middle, Supplementary Fig. 8d) in a BafA1-sensitive manner (upper right, Supplementary Fig. 8e) via mTORC1 pathway (lower right, Supplementary Fig. 8f).