Sds page sample buffer

Ovaries fragments or cells were lysed in RIPA buffer with protease inhibitor (Apexbio) and phosphatase inhibitor (Apexbio) for 10 mins and centrifuged at 12,000 g for 10 mins at 4 °C. The supernatant mixed with SDS‐PAGE sample buffer (GenStar) was denatured at 100 °C for 10 mins. Denatured protein was separated on 10–15% SDS‐PAGE gel and transferred to Polyvinylidene Fluoride membrane. Blocked with 5% non‐fat dry milk, the membranes were incubated with primary antibodies of GSDMD (Abcam, ab219800), IL1beta (Abcam, ab205924), caspase 1 (AdipoGen, AG‐20B‐0042‐C100), caspase 11 (AdipoGen, AG‐20B‐0060‐C100) and GAPDH (Affinity, AF7021) overnight at 4 °C and the corresponding secondary antibodies for 1 h at room temperature. The blots were imaged with ChemiDoc imaging system (Bio‐Rad).

Cell lysates were prepared as previously described [8 (link)]. Briefly, after the assays, the cells were lysed in cold lysis buffer (40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 1.5 mM Na₃VO₄, 0.3% CHAPS, and a mixture of protease inhibitors) for 10 min. The lysates were centrifuged at 13,000 g for 15 min at 4 °C. The supernatant was mixed with 5 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (GenStar, #E153) and boiled for 5 min. The samples were subjected to SDS-PAGE and western blotting with the indicated antibodies.

Cells were collected and lysed using reagents from a nuclear protein and cytoplasmic protein extraction kit (Beyotime Biotechnology, China) containing 1 mM protease and phosphatase inhibitor (Beyotime Biotechnology). The cell lysates were heated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Genstar, China) at 99 °C for 5 min. The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany) that were then blocked with 5% skim milk for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies, including anti-NF-κB p65, anti-phospho-NF-κB (p-NF-κB) p65, and anti-Bax (1:1000, 1:1000 and 1:1000, all Cell Signaling Technology, US), anti-Bcl-2(1:500, Santa Cruz Biotechnology, US) and anti-Gapdh (1: 50,000, Proteintech, US). The membranes were then washed with Tris-buffered saline containing Tween 20 (TBST) and incubated at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 1 h. Proteins were detected using an enhanced chemiluminescence (ECL) kit (WBLKS0500, Merck Millipore) and an automatic chemiluminescence image analysis system (Tanon, China). Quantitative analysis was performed using ImageJ software.