Non fat milk

Cells were lysed with ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The concentration of protein was quantified using a bicinchoninic acid protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Protein (60 µg/lane) was separated by 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with 5% non-fat milk (Yili, Beijing, China) in PBS for 3 h at room temperature, and then probed with rabbit anti-human versican antibody (ab19345; 1:50; Abcam, Cambridge, MA, USA) and rabbit anti-human GAPDH antibody (ab9485; 1:50; Abcam) at 4°C overnight. After washing with PBS for 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (ab6721; 1:5,000; Abcam) at room temperature for 40 min. A Chemiluminescent Substrate kit (Thermo Fisher Scientific, Inc.) was used to detect signals according to the manufacturer's protocol. The relative protein expression, represented as the density ratio vs. GAPDH, was determined using Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA).

Cells were solubilized in cold RIPA lysis buffer (Beyotime). Proteins were separated with 10% SDS-PAGE (Beyotime) and transferred onto a nitrocellulose membrane (Life Technologies), and then incubated with TBST (Beyotime) containing 5% nonfat milk (Yili, Beijing, China) at room temperature for 3 h. The membrane was then incubated with mouse anti-human TGIF2 antibody (Abcam, Cambridge, MA, USA), mouse anti-human MMP9 antibody (Abcam), and mouse anti-human GAPDH antibody (Abcam), respectively, at room temperature for 3 h. After washing three times with PBST, the membrane was incubated with the rabbit anti-mouse secondary antibody (Abcam) at room temperature for 1 h. Detection of immune complex was performed with an Pierce ECL Western Blotting KIT (Pierce, Thermo Fisher, USA), according to the manufacturer’s instruction. Image-Pro plus software 6.0 was used to analyze the expression of the protein.