Bca kit

Cells were harvested, washed and suspended in RIPA lysis buffer (Wuhan Servicebio Technology Co. Ltd.). Following 6000g centrifugation for 10 min at 4 °C, the supernatant was collected. The total protein in each sample was quantified using a bicinchoninic acid (BCA) kit (Wuhan Servicebio Technology Co. Ltd.). Equal amounts of proteins (10 µg/lane) were electrophoretically separated via 12% SDS-PAGE and transferred to a PVDF membrane (EMD Millipore). The membrane was blocked for 1 h in 5% skimmed milk dissolved in 0.1% of Tween-20 used for TBST at 4 °C and then incubated with antibodies directed against, human caspase 3 (1:1000), c-caspase 3 (1:1000), IκB (1:1000), p-IκB (1:1000), NF-κB p65 (1:1000), BCL2 (1:1000), BCL-XL (1:1000), BAX (1:1000), AIP (1:1000), p53 (1:1000), and β-actin (1:1000) at 4 °C overnight, followed by incubation with goat anti-rabbit IgG H&L (HRP) (1:1000; A0216, Beyotime Institute of Biotechnology) or goat anti-rat IgG H&L (HRP) (1:1,000; A0208, Beyotime Institute of Biotechnology) at room temperature for 1 h. The protein signals were detected using Super electrochemiluminescence (ECL) Detection Reagent (Yeasen Biotechnology Co. Ltd) by 5.2 Image Lab software (Bio-Rad Laboratories, Inc.). The bands on the membrane were quantified by normalization to β-actin.

After extraction of proteins from tissue samples and processed cell samples, the protein concentration was measured using the BCA kit (Wuhan Servicebio Technology Co. Ltd). Loading buffer (Wuhan Servicebio Technology Co. Ltd) was added, boiled at 100 °C for 10 min, and stored at –80 °C. The proteins were separated using 8%–12% SDS gel electrophoresis and transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The strip was blocked with 5% skimmed milk or 5% BSA at room temperature for 2 h, diluted at 4 °C for 12 h per the instructions of the primary antibody, incubated with the second antibody at room temperature for 2 h, and then exposed.

The vulvar tissues were homogenized in RIPA lysis buffer with protease inhibitors. The concentration of total protein was determined using the BCA kit (Servicebio, Wuhan, China). The equal amounts of denatured proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA). The membrane was sealed in skim milk and then incubated with primary antibodies against NLR family pyrin domain containing 3 (NLRP3), interferon gamma inducible protein 16 (IFI16), caspase-1 (ABclonal, Wuhan, China), ASC (Santa Cruz Biotechnology, Dallas, TX), gasdermin D (GSDMD)(CST, Danvers, MA) and secondary antibody (LI-COR, Lincoln, NE). Images of the membrane were acquired using the Odyssey infra-red imaging system (LI-COR, Lincoln, NE).