Polyvinylidene fluoride (pvdf)

After being washed with cold PBS (Gibco, Grand Island, NE), cells were lysed in 200 μl RIPA lysis buffer (Beyotime Biotechnology, Shanghai, CN) containing 1% phenylmethanesulfonyl fluoride (PMSF; New Cell & Molecular Biotech, Suzhou, CN) and were vortexed briefly and centrifuged at 20,000 g for 10 min, 4°C. The supernatant was collected and protein concentration was determined using a Bradford protein assay kit (Bio-Rad, California, USA). Equal quantities of proteins (30 μg per well) were subjected to SDS-PAGE gels (10%) and transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA, USA) membranes. PVDF membranes were incubated in Tris-Buffered Saline-Tween (TBS-T, 10 mM Tris-HCl, 100 mM NaCl, and 0.5% Tween-20) with 5% non-fat milk for 1 h and then incubated with diluted antibodies to ChemR23 (1:1,000, ab64881, Abcam, Cambridge, UK) and β-actin (1:1,000, AA128, Beyotime Biotechnology, Shanghai, CN) at 4°C overnight. After washing, membranes were blocked with 5% non-fat milk in TBS-T and probed with peroxidase-conjugated secondary antibody to rabbit or mouse (BL003A and BL001A, Biosharp, Hefei, CN), respectively (1:2,000). The immunostainings were detected with an ECL kit (Millipore Sigma, Billerica, USA) and chemiluminescence captured by Gene Tools software (Syngene, Cambridgeshire, UK).

Radioimmunoprecipitation assay buffer (Abcam) and 1% protease inhibitor (Solarbio, Beijing, China) were used to extract total protein from VCaP cells. Proteins were separated using 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis before transferring to polyvinylidene fluoride (PVDF; Millipore, Burlington, MA, USA) membrane using theconstant current wet transfer method of 350 mA for 60 min. Membranes were then blocked with 5% skimmed milk (BD Biosciences) for 2 h at 20–27 °C. After the blocking was complete, the PVDF membrane was cut according to the molecular weight of protein, and incubated with the corresponding primary antibodies anti-Bax (1: 1000, Abcam), anti-Bcl-2 (1:500, Abcam), anti-active caspase 3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), or anti-β-actin (1:2000, Cell Signaling Technology) overnight at a 4 °C. After washing in TBST, the membranes were then incubated for 2 h at20–27 °C with a horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG secondary antibody (1:10,000, Abcam). The proteins were detected using the enhanced chemiluminescence (Biomiga, San Diego, California, USA). Finally, ImageJ software (1.48v) [25 (link)] was used for semi-quantitative analysis. Each experimental operation was verified three times, with β-actin as the reference gene.

We examined whether ROP18 was correctly expressed in HEK293T cells using Western blotting analysis. Briefly, total protein was extracted using ProteinExt™ Mammalian Total Protein Extraction Kit (TRAN, China). Then, 20 µg of the extracted protein and 10 µl PageRuler™ Prestained Protein Ladder (Thermo Scientific, USA) were electrophoresed on 12% ExpressplusTM PAGE Gels (GenScript, China) under 120V and then electrotransferred to PVDF membrane (Thermo, Germany). The PVDF blotting membrane was incubated with anti-HA tag antibody (Abcam, UK) overnight at 4°C. Then, the PVDF membrane was washed three times with 1× TBS (Solarbio, China) and the PVDF membrane was incubated with secondary antibody, goat anti-mouse IgG H&L (HRP) (Abcam, UK), for 1 h at 37°C. The PVDF membrane was washed three times by 1× TBS. The ECL reagent (Solarbio, China) was used to detect the targeted protein (Solarbio, China). The Western blot image was recorded by Gel DocTM XR+ with image lab™ Software (BIO-RAD, USA).

RIPA lysis buffer (Beyotime, Shanghai, China) was bought to extract total proteins from the cells, which were further separated by using the 10% SDS-PAGE. Then, according to the molecular weight of the proteins, the target proteins were transferred onto the polyvinylidene fluoride (PVDF, Solarbio, Beijing, China) membranes. The PVDF membranes with transferred proteins were subsequently blocked by 5% blocking buffer and were probed with the primary antibodies against NLRP3 (1:1500, Abcam, Cambridge), ASC (1:2000, Abcam, Cambridge), IL-1β (1:2000, Takara, Berkeley, CA), IL-18 (1:1500, Takara, Berkeley, CA) and GAPDH (1:2000, Abcam, Cambridge) overnight at 4 °C and were incubated with the secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized by the ChemiDoc^TM XRS system (Bio-Rad, Hercules, CA) and GAPDH was used as internal control. The Image J software was employed to analyze the gray values of the protein bands.