Protease inhibitor and phosphatase inhibitor

For the C4-2 and PC-3 cell lines with or without knockdown of SRD5A2 and ITGA11, we collected the cells at log phase and lysed cells with RIPA lysis buffer (Cat. #P0013B, Beyotime, Shanghai, China) added protease inhibitor and phosphatase inhibitor (Cat. #P1405, Beyotime, Shanghai, China). Proteins (40–50 μg) were separated on 12.5% SDS/PAGE gels and then transferred onto nitrocellulose blotting membranes (GE Healthcare Life Science, Germany). Membranes were blocked with 5% bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA) for 1 h at room temperature and then incubated with appropriate dilutions of specific primary antibodies against SRD5A2 (Cat. #: DF8416, Affinity Biosciences LTD., Ohio, USA), ITGA11 (Cat. #: bs-13771R, Bioss Antibodies LTD., Massachusetts, USA), AKT1/2/3 (Cat. #: AF6216, Affinity Biosciences LTD., Ohio, USA), p-AKT1/2/3 (Ser⁴⁷³) (Cat. #: AF0016, Affinity Biosciences LTD., Ohio, USA), GAPDH (Cat. #: 1049-1-AP, Proteintech Group, Illinois, USA) overnight at 4 °C. The next day, after incubation with HRP-conjugated secondary antibodies for one hour, the membranes were visualized using an ECL system (Pierce; Thermo Fisher Scientific, Inc., USA).

Total protein was isolated using RIPA lysis buffer, supplemented with protease inhibitor and phosphatase inhibitor (Beyotime, Beijing, China). The concentration of the protein was determined by BCA Protein Assay Kit (Beyotime, P0010, China). One-fourth volume of 5 x SDS loading buffer was mixed with each extract before heating at 95°C for 5 minutes. A total of 30 μg sample of corresponding groups was added and separated by 6-15% SDS-PAGE electrophoresis, followed by transfer to PVDF membrane. After being sealed with 5% BSA at room temperature for 1 hour, the membranes were incubated with correspondent primary antibodies, which were diluted according to instructions, and stayed overnight at 4°C. Subsequently the membranes were rinsed 3 times by TBST, followed by incubation with appropriate secondary antibody. After washing 3 times by TBST, the target protein bands were exposed using ECL detection system (SmartChemi 420, Beijing, China).

Colon and liver tissues were lysed with RIPA lysis buffer supplemented with a protease inhibitor and phosphatase inhibitor (Beyotime, China) for 30 min on ice [23 (link)]. The samples were collected after centrifugation at 12,000 rpm at 4°C for 10 min, and the protein concentrations were measured by a bicinchoninic acid (BCA) protein assay kit (09282021010, Beyotime, Shanghai, China). Equal amounts of protein (40 μg) were loaded and separated on 8%~15% sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore Ltd., IPVH00010, Darmstadt, Germany). The membranes were blocked with QuickBlock™ solution (Beyotime, P0252, Shanghai, China) at room temperature for 15 min, washed in PBST (0.1% Tween-20 in PBS), and incubated with primary antibodies of ZO-1, occludin, iNOS, COX2, HO1, NQO1, Keap-1, Nrf2, gasdermin D, NLRP3, caspase 1, ASC, TLR4, MyD88, phospho-NF-κB, NF-κB, phospho-H2AX, SOD2, GPX4, and β-actin at 4°C overnight. After being washed with PBST 3 times, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) (1 : 10000) at room temperature for 1.5 h. The membrane blots were detected by an enhanced chemiluminescence (ECL) kit. All gray analyses for protein blots were performed with ImageJ software.

The total cellular protein lysate was prepared by lysing cells in RIPA lysis buffer with protease inhibitor and phosphatase inhibitor (Beyotime, China). Total cellular protein content was determined by a BCA protein assay kit (DingGuoBioTECH, China). Equal amounts of lysate proteins were separated with SDS-PAGE (12% separation gels) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% skim milk in tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated with different primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST three times, and incubated with corresponding IRDye-labeled secondary antibody at room temperature for 1 h. Finally, the protein bands were scanned using the Li-COR Odyssey system (LI-COR Biosciences, USA). Grayscale analysis of the western blots was performed using Odyssey Infrared Imaging software. At least 3 independent experiments were performed and representative results were shown.

After washing with cold PBS, cells were lysed in RIPA supplemented with PMSF (Beyotime Biotechnology, Shanghai, China) protease inhibitor and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). Lysates were adjusted to similar concentrations, mixed with 6× sample loading buffer and boiled for 5 min. The samples resolved by 4–20% SDS-PAGE gradient gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp, MA, USA). After blocked with 5% skimmed milk, the membranes were incubated with primary antibodies (YAP, p-YAP, Glut1, HKII, LDHA ERK1/2, p-ERK1/2, JNK, p-JNK, p38 and p-p38; Abcam, Cambridge, USA) (β-Actin; Zhongshan Golden Bridge Biotechnology, Beijing, China) overnight at 4 °C. Membranes were incubated with appropriate secondary antibodies (Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at room temperature, followed by detection using an ECL blotting analysis system (Bio-Rad, CA, USA).