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.

Cells or tissues were washed with cold PBS and lysed in RIPA buffer containing 1% proteinase inhibitor cocktail solution (Sigma-Aldrich) on ice for 30 min. The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific, USA) was applied to isolate nuclear fractions. Lysates were centrifuged, and protein was quantified using the BCA assay kit (Beyotime, Jiangsu, China). SDS-PAGE electrophoresis was performed on proteins from lysates and transferred them to PVDF membranes (Millipore, Bedford, MA). Membranes were incubated with primary antibody overnight at 4 °C, and then detected by Enhanced Chemiluminescence Detection Kit (BOSTER, USA). Lamin B1 was used as an endogenous control of cell nuclear fraction. All antibodies used were shown in Additional file 2: Table S2.

Total protein was extracted using RIPA buffer (Boster, Pleasanton, CA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Boston, MA) and quantified using the BCA Protein Assay (Thermo Fisher Scientific, Boston, MA) according to manufacturer’s instructions. Protein lysates were separated using 10% SDS-PAGE and transferred to PVDF membranes. Subsequently, membranes were blocked with 5% non-fat milk and incubated with UNC5B (Bioss, 11492R, 1:1000, Woburn, MA), phospho-PI3K p85α (phospho Y607) (Abcam, ab182651, 1:1000, Cambridge, MA), PI3K p85α (Abcam, ab191606, 1:1000, Cambridge, MA), phospho-Akt (Ser473) (Cell Signaling Technology, 4060S, 1:2000, Danvers, MA), Akt (Cell Signaling Technology, 2920S, 1:2000, Danvers, MA), and GAPDH (Boster, BM1985, 1:2000, Pleasanton, CA) at 4° C overnight, followed by a one-hour incubation with secondary antibodies. The signal was detected using an enhanced chemiluminescence detection kit (Boster, Pleasanton, CA). Relative protein expression was measured with ImageJ and normalized over GAPDH.