Enhanced ecl detection system

Total cellular proteins were extracted in a lysis buffer containing 1% NP-40, 0.5 M EDTA, 20 mM HEPES, 10 mM KCl, 1% glycerol and the protease and phosphatase inhibitors (Roche). 10-40 μg of protein extracts were run on 4–12% SDS-PAGE and transferred at 4°C onto a nitrocellulose membrane (0.2 micron). After 1 hour saturation at room temperature with 5% BSA in Tris-buffered saline and 0.1% Tween 20 (TBS-Tween), nitrocellulose membranes were incubated with primary antibody at 4°C overnight. Horseradish peroxidase (HRP)-conjugated goat anti–mouse or anti–rabbit (SouthernBiotech) antibodies were then incubated for 1 hour and revealed with the enhanced ECL detection system (GE Healthcare). The primary antibody against P2X7 (#APR-004) was obtained from Alomone laboratories. Primary antibodies against ACE2 (#AF933) and Spike (S) (#GTX632604) were from R&D Systems and Genetex, respectively. Primary antibody against β-Actin-HRP (#ab49900) was purchased from Abcam. Anti-NLRP3 (Cryo-2, #AG-20B-0014) was from Adipogen. P24 antibody was obtained through the NIH HIV Reagent Program (Division of AIDS, NIAID, NIH: Polyclonal Anti-Human Immunodeficiency Virus Type 1 SF2 p24 (antiserum, rabbit), ARP-4250, contributed by DAIDS/NIAID; produced by BioMolecular Technologies).

Samples, separated by a 10% SDS-PAGE, were then transferred to the polyvinylidene difluoride membrane (GE healthcare, Pittsburgh, PA, USA). The membrane was incubated with the blocking solution, containing milk, and then further washed using 0.1% and 0.3% TTBS, containing Tween-20 either 0.1% or 0.3% in Tris-buffered saline, sequentially. The anti-HIF-1α antibody (Novus Biologicals, Littleton, CO, USA) was purchased. The enhanced ECL detection system (GE healthcare) was used. Signals were detected using a Storm phosphoimager (Molecular Dynamics, Caesarea, Israel).

Protein extracts were prepared in lysis buffer (150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 1% Triton-X100, in 50 mM Tris–HCl pH 7.4), supplemented with a protease and phosphatase inhibitor cocktail (Biotool, Houston, TX, United States). Extracts were electrophoretically separated on 10% or 12% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, United States). The membranes were then blocked with 5% w/v BSA Fraction V in PBS containing 0.1% Tween-20, and subsequently incubated with anti-Phospho-Myosin Light Chain II (Ser19) (Cell Signaling Danvers, MA, United States), total Myosin Light Chain II (Cell Signaling Danvers, MA, United States), or anti-HSP90 (Santa Cruz Biotech) primary antibodies. The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA, United States) or goat anti-mouse IgG polyclonal antibody (Bio-Rad Laboratories, Inc., Hercules, CA, United States) for 1 h at room temperature. Bands were visualized with a chemiluminescence kit (Pierce, Thermo Scientific, Rockford, IL, United States), using an enhanced ECL detection system (GE Healthcare).