Transferrin receptor

For DCYTB analysis, non-reduced samples were used; other samples were reduced. Cells were lysed in NP-40 lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS) in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland) and proteins separated by SDS-PAGE. Western blots were probed with antibodies to DCYTB (Sigma-Aldrich, HPA014757), transferrin receptor (Thermo Fisher Scientific, Waltham, MA, USA, 13-6890), ferritin H [68 (link)], β-actin (Sigma-Aldrich, A3854), total FAK and P-FAK (Y925) (Cell Signaling Technology, Inc., Danvers, MA, USA, 13009 and 3284), phospho-paxillin (Cell Signaling Technology cat #2541), and paxillin (Cell Signaling Technology cat# 12065).

Proteins were isolated with RIPA buffer plus protease inhibitor cocktail (Sigma, P8340) and a tissue grinder for 5 s at 25,000 rpm (IKA; T25 digital Ultra Turrax, Staufen, Germany). Protein concentration was determined with the Pierce BCA assay kit (ThermoFisher 23225, Waltham, MA, USA). Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; ThermoFisher), transferred to PVDF membranes (Millipore, Burlington, MA, USA) and blotted with the following primary antibodies. HO-1 (in-house, need ref); TOM20 (Cell Signaling 42406, Danvers, MA, USA); total rodent oxphos antibody cocktail (Abcam ab110413, Cambridge, UK); calnexin (Enzo, ADI-SPA-860-F, Farmingdale, NY, USA); transferrin receptor (ThermoFisher 13-6800); ferritin (Abcam ab75973). Horseradish peroxidase conjugated secondary antibodies were used for visualization with Luminata Crescendo (EMD Millipore) or SuperSignal West Femto substrate (ThermoFisher) using the ChemiDoc Touch Imaging System (BioRAD, Hercules, CA, USA).

AMΦ isolated from female or male mice (WT vs. Trpml3^−/−, 2–6 months old) were isolated and cell pellets were resuspended in lysis buffer (10 mM TRIS HCl pH 8 and 0.2% SDS) supplemented with proteinases and phosphatases inhibitor (Sigma). Total cell lysis was completed by ultrasonication. Protein concentration was determined by the Bradford method. SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotting, protein visualization, membrane developing using Odyssey FC Imaging System (LI-COR) running ImageStudio software v1.0.19 and protein quantification were performed according to established protocols⁶⁷ . Sample processing controls for quantitative comparison were run on the same blots as the samples, but the blots were cut before incubation with antibodies to detect the respective protein bands. The following antibodies were used: ß-actin (Cell Signaling, 4970, 1:100 or SantaCruz, 47778, 1:1000), transferrin receptor (ThermoFisher, 13-6800, 1:500), LC3B (Novus Biologicals, 100-2220, 1:1000), Phospho-NF-κB p65 (Ser536) (Cell Signaling, 3033, 1:1000), NF-κB p65 (Cell Signaling, 6956, 1:1000), Phospho-NF-κB p105 (Ser932) (Cell Signaling, 4806, 1:1000) and NF-κB1 p105/p50 (Cell Signaling, 13586, 1:1000). Uncropped scans of all blots are supplied with the Source Data file.

For the analysis of synaptic marker expression, hippocampi from WT and Myo9a^+/- mice were dissected and homogenized in RIPA buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1% Triton X-100, protease inhibitors, pH 7.5). Ten micrograms of protein from each sample were loaded onto an acrylamide gel for western blotting_.Primary antibodies directed against the following proteins were used: Myo9a (Tü 76, WB 1:1000; Tü 78, IP 10 μg/ml; Chieregatti et al., 1998 (link); Hanley et al., 2010 (link)), Myo5a (AbCAM, 1:1000), GluA1 (Chemicon, 1:1000), GluA2 (Chemicon, 1:1000), GluA2/3 (1:2000, gift from C. Gotti), GluK2 (Prestige, 1:1000), Homer1 (Santa Cruz, 1:500), PSD95 (Neuromab, 1:20000), Synaptophysin1 (SySy, 1:5000), GAPDH (Santa Cruz, 1:1000), N-cadherin (NCAD) (AbCAM, 1:1000), PICK1 (Neuromab, 1:1000), GRIP1 (BD Transduction Laboratories, 1:2000), Tubulin (Sigma, 1:50000), Transferrin receptor (Invitrogen, 1:1000), VGAT (SySy, 1:1000), and VGLUT (SySy, 1:2000). The secondary antibodies, horseradish peroxidase- (Sigma) or IR Dyes- (LI-COR) conjugated antibodies, were used for western blots. Samples were separated using SDS-PAGE, and western blots then visualized with the Pierce ECL-Detection Kit or an Odyssey Infrared Imager (LI-COR).