Ripa solution

Total proteins in 3×10⁴ cells of each transfection group were isolated by RIPA solution (Sangon). BCA assay (Sangon) was performed to quantify protein samples. After proteins were denatured at 95°C for 10 min, proteins were separated by performing electrophoresis (12% SDS-PAGE gel). PVDF membrane was used to transfer proteins and 5% non-fat milk (PBS) was used for blocking. After that, membranes were first incubated with anti-GAPDH (1: 1000, ab37168, Abcam) and anti-TGF-β1 (1: 1000, ab92486, Abcam) rabbit primary antibodies for 18 h at 4°C. Following that, IgG-HRP (1: 1500, MBS435036, MyBioSource) goat secondary antibody was used to further incubation for 2 h at 24°C. Finally, ECL (Sigma-Aldrich, USA) was used for signal production and all data were normalized using Image J v1.48 software.

To extract total protein, transfected cells were lysed with radioimmunoprecipitation assay (RIPA) solution (Sangon Biotech Co. Ltd., Shanghai, China) supplemented with a protease inhibitor cocktail (Sangon Biotech Co. Ltd.) and phenylmethanesulfonyl fluoride (Sangon Biotech Co. Ltd.). After total protein quantification using a BCA Protein Assay Kit (Sangon Biotech Co. Ltd.), equal amounts of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h and subsequently incubated overnight at 4°C with primary antibodies against SP1 (Cat. No. b124804; Abcam, Cambridge, MA, USA) or GAPDH (Cat. No. ab128915; Abcam). The primary antibodies were used with a dilution of 1:1,000. Next, a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Cat. No. ab6721; Abcam) was incubated with the membranes at room temperature for 2 h. Finally, the protein signals were visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA).

RIPA solution (Sangon) was mixed with CSCC cells (1 mL per 10⁶ cells) to extract total proteins. A BCA kit (Sangon) was used to measure the concentrations of protein samples. Protein samples were then incubated in boiled water for 5 min to denature proteins. To separate proteins, 12% SDS-PAGE gel was used to carry out electrophoresis. After electrophoresis, proteins were transferred to PVDF membranes, followed by blocking at 23°C for 1 h in PBS containing 5% non-fat milk. The first blotting was performed using GAPDH (1:1300, ab37168, Abcam) and EPB41L3 (1:1300, ab154071, Abcam) rabbit primary antibodies (4 °C for 12 h). The second blotting was performed using HRP goat anti-rabbit (IgG) antibody (1:1000; ab6721; Abcam, 23 °C for 2 h). RapidStep™ ECL detection reagent (EMD Millipore) was dropped onto the membranes to develop signals. All gray values were normalized using Image J v1.48 software.