Anti caspase 1 sc 514

Immunofluorescence assay was performed as previously described by Donaldson [46 (link)]. CAL27 cells on glass coverslips were rinsed briefly in PBS (0.15 M NaCl, 10 mM Na₂HPO₄, pH 7.4), permeabilized in 0.2% Triton X-100/PBS, and blocked with 10% bovine albumin serum. Cells were incubated overnight (O/N) at 4 °C with primary antibodies: anti-p53 (sc-126, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-caspase-1 (sc-514, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA). After being washed in PBS, cells were incubated with secondary antibody Alexa Fluor 488 goat anti-mouse (1:1000 v/v; Molecular Probes, cat # A32723, MCR, UK) for 1 h at 37 °C. Sections were washed in PBS and 2 μg/mL 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS was added for nuclear staining. Sections were observed and photographed at 40× magnification using a Leica DM2000 microscope (Leica, Axiostar plus).

Testes placed in lysis buffer containing RIPA buffer and protease inhibitor cocktail were dispersed with a homogenizer (Thermo Fisher Scientific, USA) on ice. Then, the lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. After determination of the protein content using a Bio-Rad Protein Assay (BIO-RAD), equal amounts of extracted protein were loaded, separated via 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking with TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20) containing 5% skim milk for 1 h at 37 °C, the membrane was incubated with primary antibody at 4 °C overnight. The anti-NLRP3 antibody used was purchased from Abcam (ab205680), anti-Caspase1 (sc-514) and anti-β-actin was purchased from Santa Cruz Biotechnology. After incubation in primary antibody, the membrane was washed in TBST three times and then incubated in a horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse antibody (Santa Cruz Biotechnology) for 1 h at 37 °C. The membrane was developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Densitometric ratio analyses were performed using Image J software.