Lamp1

Whole-cell protein extracts (20–40 μg) were denatured using RIPA buffer, separated on SDS-PAGE gels, and transferred to polyvinylidene fluoride membranes. After blocking in 5% non-fat milk in Tris-buffered saline–Tween, membranes were probed overnight at 4 °C. The following antibodies were used in this study: NPC1 (Novus Biologicals, Parker, CO, USA, NB400); LDLR (Novus Biologicals, NBP1-06709); HMGCR (ThermoFisher PA5-37367); HMGCS1 (Thermo Fisher Scientific Cat# PA5-29488, RRID:AB_2546964); DHCR24 (Cell Signaling Technology Cat# 2033, RRID:AB_2091448); LAMP1 (Abcam, Cambridge, United Kingdom, Cat# ab25630, RRID: AB_470708); Phospho-S6 Kinase (Thr389) (Cell Signaling, #9205); SQSTM1/P62 (Cell Signaling #5114); LC3B (Cell Signaling, Danvers, MA #3868). Secondary antibodies were Licor IR Dye goat anti-rabbit 800 IgG (#926-32211) or goat anti-mouse 680 (#926-68070).

Primary hepatocytes were adhered onto 3T3 NIH fibroblast-coated glass coverslips and BMDCs were adhered onto poly-L-lysine-coated glass coverslips. At the end of the experimental procedures, cells were fixed in 4% paraformaldehyde solution, permeabilized in 3% BSA 0.1% saponin PBS and stained with primary Abs specific for MR-1 (AbD Serotec), EEA1 (BioConcept), LAMP-1 (Abcam), TAP1 (Santa Cruz Biotechnology), H-2Kb (BioLegend), or H-2Kb/SIINFEKL (eBioscience) followed by fluorescently labeled secondary Abs (Life Technologies) and fluorochrome-conjugated phalloidin (Life Technologies). Liver, spleen, lungs, and kidneys were harvested after perfusion of euthanized animals with HBSS (Life Technologies). Organs were fixed in 4% paraformaldehyde solution and frozen in OCT (Sakura). Ten micrometer thick sections were sliced and stained with primary Abs specific for PD-L1 or PD-L2 (eBioscience) and fluorescently labeled secondary Abs (Life Technologies) or left unstained. Samples were mounted using ProLong Gold antifade reagent with DAPI (Life Technologies), imaged with a LSM 700 inverted confocal microscope (Zeiss) and data were analyzed with ImageJ software. For flow cytometry, at the end of the experimental procedures, hepatocytes or BMDCs were washed in 2% FBS PBS and acquired on an LSR II cytometer (BD Biosciences) and data analyzed with FlowJo software (Tree Star).

Total protein was extracted from the NPCs and neurons using RIPA lysis buffer (Thermo Fisher Scientific, USA) with protease inhibitors. Protein concentrations were measured using the BCA assay (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Subsequently, 50 µg of proteins were separated by electrophoresis on SDS-PAGE and transferred onto a nitrocellulose membrane. After transfer, nitrocellulose membranes were cut to the corresponding size of the target protein for incubating with primary antibodies, without the need for stripping and reprobing procedures. Images of all replicates are provided in the Supplementary information file. Primary antibodies were incubated overnight at 4 °C at a concentration of 1:1,000 (GBA1 (Abcam (Ab55080)), LAMP1 (Abcam (Ab25630)), LC3 (Cell Signaling Technology (12,741), BiP (Cell Signaling Technology (3177)), CHOP (Cell Signaling Technology (2895)), BAX (Cell Signaling Technology (2772)), BCL-2 (Cell Signaling Technology (3498)). Following this, a secondary antibody conjugated to horseradish peroxidase at a concentration of 1:10,000 was incubated for 1 h at room temperature. The membranes were then exposed in the Bio-Rad ChemiDoc system, and the analyses were performed using Image Lab software.

For mouse cell imaging by fluorescence analysis, cells were plated over a glass coverslip lipopolysaccharide (LPS) free, in a 24 well plate, and fixed with 4% paraformaldehyde. After permeabilization, fixed cells were blocked with phosphate buffer supplemented with 10% fetal bovine serum and 0.5% Triton X-100 for 1 h at room temperature and incubated overnight at 4 °C with rabbit polyclonal antibody against Early Endosome Antigen 1 (EEA1, 1:250, Sigma-Aldrich) to localize early endosomes, mouse anti- Lysosomal-Associated Membrane Protein 1 (LAMP- 1, 1:75, Abcam) as a marker for lysosomes and rat monoclonal anti-F4/80 (1:100, Serotec), as a macrophage marker. For detection of primary antibodies, anti-rabbit Alexa Fluor 568, anti-rat Alexa Fluor 594 or anti-mouse Alexa Fluor 568 (1:1,000; Molecular Probes, Invitrogen) were incubated for 1 h at room temperature. Coverslips were mounted in Vectashield with DAPI (Vector Laboratories) and visualized under a Laser Scanning Confocal microscope Leica SP5.