Calnexin

Rabbit polyclonal antibodies were used to detect ERdj3, EDEM1, and ERp57 (Proteintech, Chicago, IL); others were to detect AAT (DAKO, Carpentaria, CA), GAPDH (Santa Cruz, Dallas, TX), Lamp1, P62, LC3B (all from Cell Signaling, Danvers, MA), and calnexin and calreticulin (both from Stressgen biotechnologies, San Diego, CA). Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands). Mouse monoclonal antibodies against BiP and AAT were purchased from BD bioscience (San Jose, CA) and R&D systems (Minneapolis, MN), respectively. Alexa Fluor 488 goat anti‐mouse IgG and Alexa Fluor 594 goat anti‐rabbit IgG were purchased from Invitrogen. Primers, Dynabeads protein A and G, Superscript VILO cDNA synthesis kit, and ERdj3 siRNA were purchased from Life Technologies, and HP DNA transfection reagent and TaqMan Universal PCR Master Mix were purchased from Roche Applied Science. Disuccinimidyl suberate (DSS) cross‐linker was purchased from Thermo Fisher scientific (Waltham, MA).

We used commercial matrix (Matrigel) for the organotypic assays. The sources of the primary antibodies used in these studies were as follows: β-catenin (BD Biosciences), cleaved-caspase 3 (Asp175) (Cell Signaling), Calnexin (Stressgen), GM130 (BD Transduction Laboratories), laminin V (Millipore), mouse AGR2 (Abnova, clone 1C3), rabbit polyclonal AGR2 antibody (Abcam), BiP antibody (Abcam), GAPDH antibody (Millipore), Transferrin antibody (Cell signalling), and O-linked-N-acetylglucosamine antibody (Abcam).
The secondary antibodies used were as follows: Alexa-Fluor-488- and Alexa-Fluor-546-conjugated anti-mouse and rabbit IgGs (Molecular Probes/Invitrogen). Reagents used in the study were diaminophenylindole (DAPI) (Sigma-Aldrich, St Louis, MO), paraformaldehyde (Sigma-Aldrich), DTT (Sigma-Aldrich) and Cycloheximide (Sigma-Aldrich).

Acutely isolated and cultured microglia or human macrophages were lysed in STET lysis buffer supplemented with protease and phosphatase inhibitors (Sigma Aldrich). Lysate protein content was quantified using Bradford assay (Biorad) according to the manufacturer´s protocol. At least 10 μg per sample were separated on a bis-tris acrylamide gel followed by western blotting either on a PVDF or nitrocellulose membrane (Millipore) using the following antibodies: Iba1 (1:1000, Wako); GFAP (1:1000, Dako); Tuj1 (1:1000, Covance); CNPase (1:1000, Abcam); NPC1 (1:1000, Abcam); NPC2 (1:1000, Sigma Aldrich); LAMP1 (1:1000, Sigma Aldrich); Cathepsin B (1:2000, R&D System); Cathepsin D (1:1000, Novus Biologicals); mouse CD68 (1:1000, AbD Serotec); human CD68 (1:500, Acris); mouse CD63 (1:1000, Abcam); mouse GRN (1:50, clone 8H10,^{85 (link)}); human GRN (1:1000, Invitrogen); TREM2 (1:10, clone 5F4^{66 (link)}); APOE (1:1000, Millipore); PLP1 (1:1000, Abcam); EGFR (1:1000, Abcam) and TGFB1 (1:1000, R&D System). Blots were developed using horseradish peroxidase-conjugated secondary antibodies (Promega) and the ECL chemiluminescence system (GE Healthcare). An antibody against Calnexin (1:1000, Stressgen) or GAPDH (1:2000, Abcam) was used as loading control. Blot densitometry quantification was performed using gel analyzer tool in ImageJ (NIH) with at least n = 3 for both human and murine samples.

Actin (Sigma, A4700) 1:400, Alpha Actinin 2 (Abcam, ab68167) 1:2000, AMPA Receptor 1 GluR1a (Alomone, AGC-004) 1:500, BIII Tubulin (Sigma, T8660) 1:2000, Calnexin (StressGen, SPA-860) 1:2000, DARPP32 (Abcam, 40801) 1:10,000, GABA(A) a1 Receptor (Alomone, AGA-001) 1:500, Glucose Transporter GLUT3 (Abcam, ab41525) 1:500, Na+/K+ ATPase (Affinity Bioreagents, MA3-915) 1:5,000, NMDA Receptor 2B GluN2B (Alomone, AGC-003) 1:500, PDE10a (Abcam, 177933) 1:2000, PSD95 (Cell Signaling, 2507S) 1:1000, SCN4B (Abcam, ab80539) 1:200, SNAP25 (BD Transduction Labs, 610366) 1:10,000, Transferrin (Thermo Fisher, 13-6800) 1:1000, VGlut1 (Synaptic Systems, 135302) 1:10,000, VGlut2 (Synaptic Systems, 135402) 1:10,000, XK (Aviva Systems Bio, ARP33809_P050) 1:1000, Huntingtin [Ab1, aa1-17, (DiFiglia et al., 1995 (link))] 1:2000, poly-Q (1C2, EMD Millipore MAB1574) 1:1000. Horseradish peroxidase secondary antibodies (Jackson Immunoresearch) were diluted 1:5000.