Ripa lysate

Kidney tissue was added to RIPA lysate (Thermo Fisher Scientific, United States) containing phosphatase inhibitor and protease, followed by being disrupted by an ultrasound. Then, the sample was centrifuged at 14,000 g at 4°C for 15 min, and the supernatant was collected. The BCA protein detection kit (Pierce, United States) was utilized for measuring the protein concentration. 20 μg protein was loaded. After 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the product was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). The membrane was blocked with 5% bovine serum albumin at 37°C for 2 h, and was incubated with primary antibodies overnight at 4°C. Antibodies included EIF4B (1/10000; ab134138; Abcam, United States), RICTOR (1/1000; ab219950; Abcam, United States), PRKCB (1/2000; ab195039; Abcam, United States), and GAPDH (1/10000; ab8245; Abcam, United States). After being washed 3 times with TBST, the membrane was incubated with HRP-labeled secondary antibody (1/2000; ab7097; Abcam, United States) for 2 h at 37°C. After being washed three times, the protein expression was detected by a gel quantitative analysis system following treatment with ECL (Solarbio, Beijing, China) with GAPDH as the reference control.

The colon tissue of mice was analyzed with RIPA lysate (ThermoFisher Inc.). Total protein in the supernatant collected was quantified by BCA method. Total protein was then subjected to SDS agarose gel electrophoresis and then transferred into PVDF membrane. The primary antibodies (Abcam, England) were added and incubated with bands overnight at 4°C. Following TBST washing, the protein bands were then incubated with the secondary antibody (Abcam, England) at room temperature for 2 h. Then, the ECL chemiluminescence reagent (ThermoFisher Inc.) was used for color development and chemiluminescence imaging.

The cultured CD4⁺ T cells under Th17/Treg differentiation conditions were lysed in RIPA lysate (Thermo Scientific, USA) plus protease inhibitors (Beyotime, China). Proteins were subjected to electrophoresis separation and then transferred to polyvinylidene fluoride membranes. The membranes were incubated with 5% milk for 1 h, followed by incubating with the GPR43 antibody (1:800, Genetex) overnight at 4°C. GAPDH (1:5,000, Elabscience) was applied as a loading control. The membranes were washed in TBST, incubated with a corresponding secondary antibody (anti-rabbit or anti-mouse IgG, 1:5,000, Elabscience) for 1 h, and washed again by TBST. The protein bands were visualized by an enhanced chemiluminescence (ECL) system (ChemiScope 5600; Hengmei Technology, China).

BEAS-2B cells and LC cells were collected, and total protein was extracted using RIPA lysate (Thermo Fisher Scientific, Waltham, MA, USA). BCA kit (Beyotime, Shanghai, China) was used to determine the concentration of total cellular protein. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis was used to separate protein, and then the protein was transferred to polyvinylidene fluoride (PVDF) membrane. After blocking the PVDF membrane with 5% bovine serum albumin, the PVDF membrane was incubated with diluted primary and biotin-coupled secondary antibodies. Enhanced chemiluminescence reagent (ThermoFisher Scientific, Waltham, MA, USA) was applied for development, and observation and photography were performed by a ChemiDoc XRS + gel imaging system (Bio-Rad, Hercules, CA, USA). GAPDH was used as the internal reference. Analysis of the target protein was performed by Image J software (National Institutes of Health, Bethesda, MD, USA). All antibodies above were bought from Abcam (Cambridge, UK). Antibody information was shown in Table 2.

Colon samples were homogenised in RIPA lysate (Thermo Fisher Scientific, USA), and the supernatant was collected for assessments of pro-inflammatory cytokines (n = 4 for each group). TNF and IL-6 were detected using the commercial ELISA kits (Biolegend, USA), according to the instruction from the manufacturer. The final results were represented as the ratio of cytokine concentration to total protein content, which was determined using a BCA reagent kit (Beyotime Institute of Biotechnology, China).

Firstly, the total protein was extracted by RIPA lysate (Thermo Fisher Scientific, USA) and analyzed by BCA protein quantitative kit (Thermo Fisher Scientific, USA). After that, the proteins were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membrane (Washington, New York). At room temperature, the membrane was sealed in 5% bovine serum albumin (BSA) for 1 h, and then incubated with primary antibody at 4°C overnight. The antibody was purchased from The antibodies were purchased from Cell Signaling Technology and were used at manufacturer-recommended dilutions. After that, the blot was incubated with HRP-conjugated secondary antibody (Santa Cruz, USA) at room temperature for 1 h. Finally, ECL kit (Thermo Fisher Scientific, USA) was used to observe the protein bands. GAPDH was used as internal reference.