Sds reducing buffer

Total protein was extracted using Sodium dodecyl sulfate (SDS) reducing buffer (Biorad, CA, USA) and separated on 8–12% gradient SDS polyacrylamide gels and transferred on Polyvinylidene difluoride (PVDF) membranes (Biorad, CA, USA). Membranes were blocked with 5% non-fat dry milk for 30 min at room temperature, followed by overnight incubation with a primary antibody against caspase-1 (1:300, Santa Cruze) at 4 °C. Membranes were washed with PBS with 0.1% Tween 20 and incubated for one hour at room temperature with a secondary antibody (goat polyclonal anti-mouse IgG, ab205719, 1:3000) for one hour. Membranes were stained using anti-beta-actin (1:4000, A01546, GenScript) antibody to normalize protein expression for total protein and cytoplasmic fraction. Membranes were stained with anti-lamin b1 (1:300, sc-20682, Santa Cruz) antibody and secondary human, bovine, horse, horseradish peroxidase-conjugated goat anti-mouse IgGs (H&L) (A106PS; American Qualex) to normalize protein expression for nuclear proteins. Western blot results were visualized using Clarity Western ECL reagents (Biorad, CA, USA) and a ChemiDoc XRS + (Biorad, CA, USA).

Total protein extracts were prepared using Sodium dodecyl sulfate (SDS) reducing buffer (Biorad, CA, USA), separated on 8–12% gradient SDS polyacrylamide gels and transferred on Polyvinylidene difluoride (PVDF) membranes (Biorad, CA, USA). Membranes were blocked [Tris-buffered saline (TBS), 0.1% Tween 20, 5% BSA] for 1 h followed by overnight incubation with the monoclonal rabbit anti-human NLRP3 (1:300, Invitrogen, IL, USA) antibody at 4°C. Membranes were washed with TBS and 0.1% Tween 20 and incubated for 1 h at room temperature with anti-rabbit IgG (1:1,000, Santa Cruz Biotechnology, Germany) and mouse anti-human Actin Beta-HRP conjugated (1:1,000, Sigma) antibodies. Western blot results were visualized using Clarity Western ECL reagents (Biorad, CA, USA) and a ChemiDoc XRS + (Biorad, CA, USA). Protein levels were quantified using NIH ImageJ software version 1.52a.