Enhanced chemiluminescence detection kit

Total proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime, China) or the Mitochondrial Protein Extraction kit (BOSTER, China), and the protein concentration was determined using the BCA Protein Quantification Kit (BOSTER, China). The proteins were subjected to 10% SDS-PAGE and then transferred to a PVDF membrane, blocked with TBST containing 5% skim milk, and probed with Bax (Abclonal, China), Caspase-3 (Abclonal, China), Caspase-9 (Abclonal, China), and cytochrome c (Cytc, Abclonal, China) monoclonal antibodies. The protein bands were visualised using an enhanced chemiluminescence detection kit (BOSTER, China) and photographed using a gel imager (Bio-Rad, America).

Proteins were extracted with the cell and tissue total protein extraction kit (cat# KC415, Shanghai Kang Cheng Bioengineering Co., Ltd., Shanghai, China). Briefly, the protein concentration was quantified with a bicinchoninic acid (BCA) protein assay kit (cat# P0010, Beyotime, Nantong, China), and then the protein samples were diluted to the same concentration with a 5 × loading buffer (cat# P0015L, Beyotime) and double-steamed water (cat# A500197-0500, Shanghai Sangon Biotech Co., Ltd., Shanghai, China) and boiled in a metal bath at 100 °C for 10 min. Equal amounts of the proteins were loaded and separated using 10% or 15% SDS-PAGE, and then electro-transferred to polyvinylidene difluoride membranes. The membranes were blocked at room temperature for 1.5 h with 5% non-fat milk or 5% BSA, and then incubated overnight at 4 °C with the following corresponding primary antibodies: NLRP3 (1:1000, cat# 19771-1-AP, Proteintech, Wuhan, China), caspase-1 (1:1000, cat# 22915-1-AP, Proteintech), IL-1β (1:1000, cat# Ab-AF5103, Affinity, Denber, CO, USA), IL-18 (1:1000, cat# ab71495, Abcam), N-domain of GSDMD (GSDMD-N, 1:800, cat# ab219800, Abcam), ASC (1:600, cat# sc-271054, Santa Cruz Biotechnology, Stanta Cruz, CA, USA), and GAPDH (1:1000, cat# 60004-1-Ig, Proteintech). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000, cat# GB23303, Servicebio, Wuhan, China) or goat anti-mouse IgG secondary antibodies (1:3000, cat# GB23301, Servicebio) at room temperature for 2 h. The protein bands were visualized with an enhanced chemiluminescence detection kit (Boster Biological Technology, Ltd., Wuhan, China) and a chemiluminescence imaging analyzer. The blot images were processed and quantified with ImageJ software. GAPDH served as an internal control.

The lysis of cervical cancer cell was achieved in RIPA buffer (Solarbio Science, Beijing, China) following the instruction provided by the manufacturer. The protein concentrations in the extracts were determined using BCA Protein Assay Kit (Takara). The extracts containing 30 μg of total proteins were mixed with commercial 5× protein loading buffer (Bosterbio, Pleasanton, CA, USA) and then subjected to 10% SDS/PAGE. After the proteins electroblotted to PVDF membrane (Bosterbio), the membrane was blocked with 5% nonfat milk in Tris‐buffered saline with Tween‐20 (TBST) buffer. The membrane was then incubated with mouse anti‐PTEN or anti‐GAPDH antibody (1 : 200 dilution; Bosterbio) at 4 °C overnight. The membrane which was bound by anti‐PTEN or anti‐GAPDH antibody was washed three times with TBST buffer and then incubated with horseradish peroxidase‐conjugated rabbit anti‐mouse IgM (1 : 2000 dilution; Bosterbio) for 2 h. After that, the membrane was washed three times with TBST buffer once again. The protein contents were shown using an enhanced chemiluminescence detection kit (Bosterbio) and analysed using alphaview sa software (ProteinSimple, Santa Clara, CA, USA). The band intensities of GADPH proteins were utilized as internal controls for the normalization of PTEN protein levels.

The regenerating liver tissues stored in liquid nitrogen were ground into fine
powder and then suspended in extraction buffer (7 M urea, 2 M thiourea, 4%
CHAPS). Next, the suspension was vortex-mixed for 1 h at 4 °C, and subsequently
centrifuged at 20,000 x g for 1 h at 4 °C. The supernatants
were collected and stored at –80 °C for further use. The protein concentration
was assessed with the commercial RC DC Protein Assay Kit according
to the manufacturer’s instructions (BIO-RAD, USA). Protein samples, 50 μg, were
separated by electrophoresis on 12% SDS/PAGE gels and subsequently
electrophoretically transferred to polyvinylidene difluoride membranes
(Millipore). The membranes were blocked with 5% non-fat milk, washed, and
subsequently probed with antibodies against 14-3-3β/α, γ, ζ/δ, σ, ε, η, τ/θ (all
1:1000) and GAPDH (1:2000) (Sangon Biotech Co. Ltd., Shanghai, China)overnight
at 4 °C. After being washed, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies (Sangon Biotech Co. Ltd., Shanghai,
China), detected with an enhanced chemiluminescence detection kit (Boster
Corporation, China) and then imaged in an ImageQuant LAS 4000 mini (GE
Healthcare Bio-Sciences Corporation) system.