Sc 28385

Fluorescence cell imaging procedures were performed as follows^{7 (link)}. Cells were fixed with 4% paraformaldehyde at room temperature for 20 min, and were permeabilized with 0.1% Triton X-100/10% FBS/PBS for 1 h. To detect ANXA2, γ-H2AX, or COL6, cells were stained using a monoclonal mouse anti-human ANXA2 antibody (200 µg/mL, sc-28385, Santa Cruz Biotechnology, Dallas, TX) at a dilution of 1:50, a mouse monoclonal anti-human phospho-histone H2AX (Ser139) antibody at a dilution of 1:250, and a polyclonal rabbit anti-human COL6 antibody at a dilution of 1:2500, respectively. After staining with each primary antibody, cells were stained using the following secondary antibodies: the goat anti-mouse IgG labeled with Alexa Fluor 594 and goat anti-rabbit IgG labeled with Alexa Fluor 488 at a dilution of 1:1000. Nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (P36934, Invitrogen). Fluorescence images were detected under fluorescent microscopes (BZX700, Keyence, Osaka, Japan, BX-51, Olympus, or IX71, Olympus). The number of cells that were positive for γ-H2AX in the nucleus or COL6 in the cytoplasm was determined using Win ROOF 2015 software. The signal intensity of ANXA2 (Fig. 5b) was measured by Image J^{31 (link)}.

Western blotting was performed as previously described [32 (link)].The antibodies used were as follows: anti-FGF19 (A6589, ABclonal, China, 1:500), anti-TRIM21 (12108–1-AP, Proteintech, China, 1:1000), anti-ANXA2 (sc-28385, Santa Cruz Biotechnology, Texas, USA, 1:1000), anti-Tubulin(FD0064, Fude bio, China, 1:1000), anti-p-PI3K (A0982, ABclonal, China, 1:500), anti-AKT1 (A11016, ABclonal, China, 1:1000), anti-p-AKT1 (AP0140, ABclonal, China, 1:1000), anti-mTOR (A11355, ABclonal, China, 1:500), and anti-p-mTOR (ab109268, Abcam, UK, 1:1000).