Ecl plus detection reagent

Cells were lysed with RIPA lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics) and fractionated using the NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific). Supernatants were filtered and concentrated by trichloroacetic acid precipitation, followed by aceton desiccation. Proteins were run on SDS-PAGE (4–12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (GE Healthcare) blocked with 5% bovine serum albumin (BSA) or non-fat dry milk (NFDM), incubated with primary antibody: rabbit anti-IL-1 beta (1:2,500 in 5% NFDM, ab9722, Abcam), rabbit anti-ASC (1:200 in 5% BSA, sc-22514-R, Santa Cruz), mouse anti-NLRP3/NALP3 (1:1,000 in 5% milk, Cryo-2, Adipogen) or rabbit anti-MMP7 (1:200 in 5% BSA, ab4044, Abcam), washed with PBS tween 0.1% and incubated with secondary antibodies in 5% NFDM (goat anti rabbit-HRP or goat anti-mouse-HRP, Cell Signaling). Bands were imaged using ECL Plus detection reagent (GE Health Care) and quantified using ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loading control.

After infection, cells were lysed with NP-40 lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics). Total cellular proteins were run on SDS–polyacrylamide gel electrophoresis (4 to 12% bis-tris gels; Invitrogen), blotted onto poly-vinylidene difluoride membranes (GE Healthcare), blocked with 5% non-fat dry milk (NFDM), and incubated with rabbit anti–IRF-7 (1:300, ab62505, Abcam, Cambridge, United Kingdom) rabbit anti-TRPC1 (1:500, #ACC-010, Alomone Labs) and rabbit anti-TRPV6 (1:500, #orb158655, Biorbyt) antibodies. The blots were washed with PBS Tween 0.1% (PBST) and incubated with HRP-linked secondary antibodies in 5% NFDM (1:4,000, goat anti-rabbit- horseradish peroxidase (HRP), #7074, Cell Signaling). The anti-β-actin (1:4,000 in 5% NFDM, #A1978, Sigma-Aldrich) followed by rabbit anti-mouse Immunoglobulins HRP-linked (1:4,000 in 5% NFDM, P0260, Dako) was used as loading control. The blots were washed with PBST and developed with ECL Plus detection reagent (GE Healthcare). Blots were imaged using the Bio-Rad ChemiDoc System (Bio-Rad) and quantification of densitometry of bands was done using the ImageJ software (NIH).

Samples were boiled in Laemmli buffer (working concentration 50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol (DTT), 2% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue, 10% glycerol). Proteins were resolved on NuPAGE pre-cast Tris-Glycine gels (Invitrogen, U.K.) prior to transfer onto nitrocellulose at 70 mA per gel for 40–60 min. Membranes were blocked in 5% non-fat milk powder/Tris-buffered saline/0.1% Tween-20 (TBST; 50 mM Tris, 150 mM NaCl, 0.1% Tween-20) with gentle agitation for one hour at room temperature (RT) or overnight at 4°C. Primary antibody was added to the membranes at the appropriate dilution in 5% Marvel/TBST with gentle agitation for 1.5 h at RT or overnight at 4°C. Membranes were washed 3 times in 1× TBST for 10 minutes. The appropriate HRP-conjugated secondary antibody (DAKO) was added to the membranes in 5% non-fat milk powder/TBST and incubated at room temperature for 45 min with gentle agitation. Membranes were washed 3 times in 1× TBST for 10 min. Bands were then visualised with ECL Plus detection reagent (GE Lifesciences, Little Chalfont, U.K.) using an AGFA Curix developer.

Total protein lysates were prepared using lysis buffer containing 1 mM protease inhibitor cocktail (Roche), 25 μg proteins loaded onto a 10% Bis-Tris NuPage gel (Invitrogen) and transferred onto a nitrocellulose membrane (Invitrogen). The membrane was incubated with primary antibodies against phospho-p44/42 MAPK and p44/p42 MAPK (Extracellular-signal-regulated kinase (ERK))(Cell Signaling) overnight at 4 °C, followed by secondary, horseradish peroxidase-conjugated antibody (goat anti rabbit IgG (Southern Biotech). Blots were developed using ECL plus Detection Reagent (GE Healthcare) and visualized on a Chemidoc MP System (BioRad). Data were analysed using Image Lab 4.1 software (BioRad).

Adult WT and conditional KO male or female whole brains or cortical regions were sonicated in lysis buffer (50 mM HEPES pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF in the presence of antipain, pepstatin and leupeptin) then left to rotate at 4°C for 1 h. Membranes were pelleted by centrifugation at 14000 g for 15 min at 4°C. Protein content of the supernatant was assayed by BioRad protein assay. Samples were then suspended in 3X protein sample buffer and analyzed by SDS-PAGE and western blotting. Briefly, protein samples were separated by SDS-PAGE on 10% Tris-Glycine gels and blotted onto nitrocellulose membranes (GE Healthcare Bio-Sciences). Membranes were blocked for 1 h in milk (PBS, 0.05% Tween, 4% milk), incubated in primary antibodies diluted with milk overnight at 4°C before incubation in an appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The blots were developed with an ECL-Plus detection reagent (GE Healthcare Bio-Sciences). Densitometric analysis was performed in ImageJ (NIH).

For Western blot analysis, 10–20 μg of protein were subjected to electrophoresis on 10 or 12% SDS-PAGE gels and blotted onto polyvinyl-idenedifluoride (PVDF) membranes. Membranes were treated with antibodies using standard Western blot techniques. The ECL Plus Detection Reagent (GE Healthcare, Chalfont St Giles. UK) was used for chemiluminescent detection, and membranes were analyzed using X-Omat LS films (Carestream, Rochester, NY) in a Curix 60 developer (Agfa, Mortsel, Belgium). Densitometry analysis was performed using the ImageLab Software (Biorad, Hercules, CA).
Antibodies used were: anti-LMP2 (Abcam, Cambridge, UK), anti-LMP7 (Abcam), anti-beta5 (Santa Cruz Biotechnology, Dallas, TX), anti-Tbp1 (Bethyl Laboratories, Montgomery, TX), anti-PSMD7 (Abcam), anti-PSMD11 (Novus Biologicals, Littleton, CO), anti-Alpha1–7 (Abcam), HRP conjugated anti-GAPDH (Cell Signaling, Cambridge, UK), and HRP conjugated anti-β-Actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies used were HRP conjugated goat anti-mouse IgG, and HRP conjugated goat anti-rabbit IgG (GE Healthcare).

Cells were lysed with NP-40 lysis buffer or by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Roche Diagnostics). 7 µg of proteins were run on SDS-PAGE (4–12% Bis-Tris gels, Invitrogen) and blotted onto PVDF membranes (GE Healthcare), blocked (5% NFDM) and stained using anti-NK1R, anti-Substance-P, anti NLRP-3 or anti-ASC primary antibodies. β-actin or GAPDH served as the loading control. Bands were imaged using ECL plus detection reagent (GE Health Care) and were quantified by ImageJ.