Anti mouse caspase 1 p10

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.

Mouse lung tissue or MLVEC were homogenized or lysed (1 × 10⁶ cells per ml) in lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 20 mM PMSF). The supernatants were quantified, and 600 μg total protein for each sample was then immunoprecipitated with anti-ASC Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-TXNIP Ab (MBL International, Ottawa, IL, USA). The immunoprecipitated proteins were separated on a 10% SDS-PAGE gel and then electroblotted onto polyvinylidene difluoride membrane and blocked for 1 h at room temperature with Odyssey Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA). Nlrp3 was detected by probing the membranes with anti-Nlrp3 Ab (Santa Cruz Biotechnologies) at 1 : 500 dilution and detected with fluorescent secondary antibody (LI-COR Biotechnology) following the manufacturer's instructions. Blots were then stripped and reprobed with anti-ASC Ab or anti-TXNIP Ab and again detected with fluorescent secondary antibody (LI-COR Biotechnology). Caspase-1 cleavage in the lung tissue or MLVEC was measured by detecting its p10 fragment by western blot using rabbit polyclonal anti-mouse caspase-1 p10 (Santa Cruz Biotechnologies). TXNIP protein in MLVEC was detected by western blot using anti-TXNIP Ab.