Lamtor4

SVs (10–20 µg) from each animal were loaded into 4–12% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen) and immunodetection. Nonfat milk (5%) was used to block nonspecific antibody binding (Thermo Fisher Scientific, Waltham, MA, USA). After blocking, membranes were incubated overnight at 4 °C with a primary antibody. Primary antibodies included GAPDH (Invitrogen), PSD95 (Invitrogen), GFAP (Sigma-Aldrich, St. Louis, MO, USA), SYP (ThermoFisher), VGLUT1 (Santa Cruz Biotech, Dallas, TX, USA), and SNAP25 (Synaptic Systems, Goettingen, Germany). MEGF8 (Bioworld Technology, St. Louis Park, MN, USA) and LAMTOR4 (Cell Signaling, Danvers, MA, USA) were additionally selected from the proteomic analysis for post-validation. Secondary antibodies were HRP-conjugated anti-rabbit IgG (Thermo Scientific) and HRP-conjugated anti-mouse IgG (Thermo Scientific). Primary and secondary antibody dilutions were done according to the manufacturer’s suggestion and are shown in Supplementary Table S1. Blots were developed using Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

For immunohistochemistry experiments, antibodies against Tfeb/TFEB (Cat. No. A303-673A, Bethyl Laboratories), cathepsin B (CTSB; Cat. No. 31718, Cell Signaling Technologies), cathepsin D (CTSD; Cat. No. 2284, Cell Signaling Technologies), transcription factor E3 (TFE3; Cat. No. PA5-54909, Invitrogen), phospho-rpS6:Ser^235/236 (Cat. No. 4858, Cell Signaling Technologies), and glycoprotein nonmetastatic melanoma protein B (GPNMB; Cat. No. PA5-42585, Thermo Fisher Scientific) were used. For immunocytochemistry experiments, TFEB (Cat. No. PA1-31552, Invitrogen), late endosomal/lysosomal adaptor and MAPK and mTOR activator 4 (LAMTOR4; Cat. No. 13140, Cell Signaling Technologies), light chain 3B (LC3B; Cat. No. ab192890, Abcam), and sequestosome 1 (SQSTM1)/p62 (Cat. No. 23214, Cell Signaling Technologies) were used. For three-dimensional (3-D) cyst culture experiments, TFEB (Cat. No. A303-673A, Bethyl Laboratories) was used.
Fluorescent-conjugated secondary antibodies were purchased from Abcam (Gt α rabbit DyLight 488, Cat. No. ab96899; Gt α mouse DyLight 488, Cat. No. ab96879; Gt α mouse DyLight 550, Cat. No. ab96872; and Gt α rabbit DyLight 550, Cat. No. ab96884).

We performed western blotting analysis as previously described (17 (link)). Briefly, 30 µg of detergent soluble S1 protein was loaded to measure the relative abundance of the protein target. Protein samples were loaded onto the Novex™ 4 to 20% Tris-Glycine Plus 20-well Midi Protein Gels (1.0 mm, Thermo Fisher Scientific, Invitrogen™, #WXP42020BOXA). For a given probing target, all groups of mice were loaded to the same gel including resolving, transferring, and exposure. The following primary antibodies were used: Anti-Liver Arginase (abcam, #ab124917), LAMTOR2 (Cell Signaling Technologies, #8145), LAMTOR3 (Cell Signaling Technologies, #8168), LAMTOR4 (Cell Signaling Technologies, #12284), RagA (Cell Signaling Technologies, #4357), and anti-β-actin antibody (Sigma-Aldrich, #A5441). Densitometric analysis was performed using AlphaEase software (Alpha Innoch, CA, US).