Radioimmunoprecipitation assay (ripa)

All transfected cells were seeded into 6-well tissue culture plates (1–2×10⁶) at 37°C and lysed using 200 µl protein (RIPA; Sangon Biotech Co., Ltd., Shanghai, China). The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Sangon Biotech Co., Ltd.). The proteins were separated using 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). Following blocking of the membranes using 5% nonfat milk in TBST, the membranes were incubated with anti-p-SMAD3 (#9520; dilution 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-TGF-β (#3709; dilution, 1:2,000; Cell Signaling Technology Inc.) and β-actin (#4970; dilution 1:2,000; Cell Signaling Technology, Inc.) antibodies at 4°C overnight. Following washing with TBST, the membranes were incubated with a goat anti-rabbit secondary antibody (#A16110; 1:5,000; Pierce; Thermo Fisher Scientific, Inc.) at 37°C for 1 h, and the blots were detected using an enhanced chemiluminescent reagent (#G-21234; Pierce; Thermo Fisher Scientific, Inc.) and quantified using Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Total protein was isolated from tissues or cells by RIPA (Sangon, Shanghai, China). Equal amounts of each sample were loaded and separated by 10% SDS-PAGE. Proteins were transferred from gel to PVDF membranes (Millipore, USA) and blocked with 5% silk milk for 1 h. at room temperature. The membranes were then incubated with specific primary antibodies: anti-JUND (1:1000, CST, Beijing); anti-β-Actin (1:2500, CST, Beijing) overnight at 4°C. Subsequently, the membranes were incubated with HRP-conjugated antibody for 2 h at normal temperature. The blot signals were washed by TBST and visualized with an ECL Kit (Solarbio, Beijing, China). Images were taken by an ImageQuant LAS4000 Biomolecular Imager (GE Healthcare).

HeLa cells incubated at 48 h in each group were lapped with radioimmunoprecipitation buffer (RIPA; Sangon Biotech) containing phenylmethanesufonyl fluoride (PMSF; Sigma-Aldrich), followed by centrifugation at 12,000 rpm for 10 min at 4°C. Supernatant was collected for the detection of protein concentrations using BCA protein assay kit (Pierce, Rockford, IL, USA). For Western blot analysis (17 ), 30 µg protein per cell lysate was subjected onto a 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidencefluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked in Tris-buffered saline Tween (TBST) containing 5% nonfat milk for 1 h. After that, the membrane was incubated with rabbit anti-human antibodies (mTOR, PI3K, P70, 1:100 dilution; Invitrogen) and then overnight at 4°C. The membrane was then incubated with horseradish peroxidase-labeled goat anti-rat secondary antibody (1:1,000 dilution) at room temperature for 1 h. Finally, PVDF membrane was washed with 1× TBST buffer for 10 min three times. Detection was conducted using the development of X-ray after chromogenic substrate with an enhanced chemiluminescence (CEL) method. Additionally, GAPDH served as the internal control.

Cells were lysed with radioimmunoprecipitation assay (RIPA; Sangon Biotech, P.R. China) containing phenylmethanesufonyl fluoride (PMSF; Sigma-Aldrich). After centrifugation at 12,000 bmp for 10 min, the supernatant was collected and the protein concentration was detected by the BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of extracts was subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany), and probed with primary antibodies to INPP4A, GSK3β, Bax, Bcl-2, pro-caspase 3, cleaved-caspase 3, c-Myc, cyclin D1, P27, β-catenin, p-β-catenin, and GAPDH (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Followed by exposure to horseradish peroxidase-labeled appropriate secondary antibody (1:5,000 dilution; Santa Cruz Biotechnology) for 2 h, the blots were incubated with a chemiluminescent substrate kit (Pierce) and then detected using the enhanced chemiluminescence (ECL) method. GAPDH served as the internal control.

Cells were washed with ice-cold PBS and lysed with radioimmunoprecipitation (RIPA; Sangon Biotech) solution containing phenylmethanesufonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was measured using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Then an equal amount of protein per lane was separated on a 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bands were then transferred onto a polyvinylidencefluoride (PVDF) membrane (Millipore). After being blocked in Tris-buffered saline Tween (TBST) containing 5% nonfat milk, the membrane was incubated with primary antibodies overnight at 4°C. GAPDH served as the internal control. The antibody against CSTB was purchased from Novus International Inc. (Saint Charles, MO, USA). Antibodies against p-PI3K (pY-458), p-Akt (pS-473), p-mTOR (pS-2448), and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Then the membrane was incubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. After being washed three times with 1× TBST buffer, the bands were detected with a chromogenic substrate using the enhanced chemiluminescence (ECL) method and further quantified by the ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).