Asc ab

Primary antibodies for cell staining and western blotting: ASC Ab (Santa Cruz Biotechnologies), rabbit polyclonal anti-mouse HMGB1 antibody (Abcam, Cambridge, MA, USA), rabbit polyclonal anti-mouse caspase-1 p10 (Santa Cruz Biotechnologies), and GAPDH (D16H11) XP Rabbit mAb (Cell Signaling Technology). Secondary antibodies including Alexa Fluor 488-conjugated anti-mouse IgG, Cy5-conjugated anti-mouse IgG, Alexa Fluor 488-conjugated anti-rabbit IgG, and Cy3-conjugated anti-rabbit IgG were provided by the Center for Biologic Imaging, University of Pittsburgh Medicine Center. In Situ Cell Death Detection Kit, TMR red (TUNEL) was purchased from Roche (Indianapolis, IN, USA). Annexin-V detection kit was purchased from BD Biosciences. iScript™ Reverse Transcription Supermix and iTaq™ Universal SYBR® Green Supermix were purchased from Bio-Rad. Phorbol 12-myristate 13-acetate(PMA) was from Sigma-Aldrich.

Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4 °C for 20 min. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF (poly(vinylidene fluoride)) membrane. The membrane was blocked in 5% fat-free milk in PBST (PBS with 0.05% Tween-20), followed by incubation overnight with the following primary antibodies diluted in PBST: JNK Ab, p-JNK Ab, p38 Ab, p-p38 Ab, ASC Ab, procaspase-1 Ab (diluted to 1:1000, all from Santa Cruz Biotech (Dallas, TX, USA)), p65 Ab (Genetex, Taiwan) and NLRP3 Ab (Adipogen, San Diego, CA, USA). The primary antibodies were removed, and the membrane was washed extensively in PBST. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:20,000, Santa Cruz Biotech) was performed at room temperature for 2 h. The membrane was washed extensively in PBST to remove any excess secondary antibodies, and the blot was visualized with enhanced chemiluminescence reagent (GE Healthcare, South Jakarta, Indonesia).