Ii system

The harvested cells were washed with phosphate buffered saline and lysed with RIPA lysis buffer (Boster, China), which was obtained total cellular protein. The protein concentration was tested by BCA Protein Assay kit (Boster, China). In order to detect the levels of protein expression, the protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a poly-vinylidene fluoride (PVDF) membrane through Bio-Rad II System. The membrane was blocked by 5% skim milk powder at room temperature for 1 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies included caspase-3(Abcam, USA; ab4051), cleaved-caspase-3 (Cell Signaling Technology, USA; #9661), Bax (Abcam, USA; ab182733), Bcl-2 (Abcam, USA; ab692), IL-6R (Boster, China; A01425-1), β-actin (Bioss, China; bs-0061R), and GAPDH (Abcam, USA; ab8245) at 1:500 dilutions. Subsequently, membranes were washed with TBST and incubated with a 1:5000 dilution of HRP-conjugated secondary antibodies (Abcam, USA) for 1 h at room temperature. The protein bands were visualized by ChemiDocTMMP imaging system (Bio-Rad, USA). The GAPDH and β-actin were used as inner loading control, respectively. The gray value was analyzed by Image-ProPlus software.

Cells were lysed in RIPA lysis buffer. Total cell protein was extracted and quantified. Blots were prepared using polyacrylamide gels and nitrocellulose membranes using a Bio-Rad II system. The antibodies used were rabbit anti-mouse NLRP3 (CST), rabbit anti-mouse caspase-1 (ImmunoWay), rabbit anti-mouse XO (Abcam), rabbit anti-mouse GAPDH (Bioworld), and goat anti-rabbit conjugated to horseradish peroxidase-2 (Zhongshan Biological Company, Beijing, China). The images were obtained and analyzed by using an ECL developer and gel image analysis system.

Cells specimens were lysed using RIPA lysis buffer (Boster, Wuhan, China). Subsequently, protein samples (20 μg) were separated by 10% SDS-PAGE gel and then transferred onto a PVDF membrane (Millipore, MA, USA) via a Bio-Rad II System (Bio-Rad Laboratories, Inc.). Then the membranes were blocked with 5% skim milk powder at room temperature for 1 h and incubated with a rabbit monoclonal antibody against STAB2 and β-actin (1:500; Cell Signaling Technology, MA, USA) and goat anti-rabbit IgG. Protein bands were detected by an ECL chemiluminescence kit (Millipore, MA, USA) according to the manufacturer's instructions. Protein levels were calculated relative to β-actin.