Primary antibodies specific for col1a1

H₉C₂ cells were plated in 35 mm dishes, washed with PBS and then fixed with 4% paraformaldehyde for 20 mins at room temperature. After being washed thrice with PBS, the H₉C₂ cells were blocked in 5% goat serum for 1 h. The cells were subsequently incubated with primary antibodies specific for Col1A1 (1:400, Abcam) overnight at 4°C. On the next day, the H₉C₂ cells were washed with PBS and incubated with fluorescence-conjugated secondary antibodies (Beyotime, China) followed by DAPI. Images were captured under a fluorescence microscope.

Total protein was extracted from cells with RIPA lysis buffer (Beyotime, P0013B, China) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and various inhibitors (sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc.), and then quantified with a BCA Protein Assay Kit (Thermo Scientific, USA). A 30 µg denatured protein sample was separated by 10–12% SDS–PAGE and was then transferred to PVDF membranes (Millipore). After blocking in 5% nonfat milk for 2 h at room temperature, the membranes were incubated with primary antibodies specific for COL1A1 (1:1000, Abcam, UK), PI3K (1:500, Abclonal Technology, USA), p-PI3K (1:500, Abclonal Technology, USA), AKT (1:500, Abclonal Technology, USA), and p-AKT (1:500, Abclonal Technology, USA) overnight at 4 °C. Thereafter, incubation with the secondary antibody was conducted for an additional 1 h at room temperature. Finally, an ECL reagent was employed for protein visualization (Millipore, USA). GAPDH was employed as the internal control.