Kindlin 2

Immunofluorescence staining was performed as described previously^{36 (link)}. Briefly, cells cultured on the glass coverslips or tissue sections were stained with following antibodies: Kindlin-2 (Proteintech, 1:100 dilution), Fibronectin (FN) (Abcam, 1:100 dilution), α-SMA (Abcam, 1:100 dilution), Smad2/3 (Cell Signaling Technology, 1:100 dilution), p-Smad2 (Cell Signaling Technology, 1:100 dilution), and p-Smad3 (Cell Signaling Technology, 1:100 dilution) overnight at 4 °C, followed by incubation with FITC or PE-conjugated secondary antibodies (Invitrogen). The nuclei were the stained with DAPI. All immunofluorescence was then visualized by Carl Zeiss MicroImaging system (Carl Zeiss, Germany).

Whole-cell extracts were prepared with 1% sodium dodecyl sulfate (SDS) lysis buffer with freshly added 1% proteinase inhibitor cocktail (MCE, HY-K0010) and 1mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, 32998-6). Protein concentrations were measured using the Pierce^TM BCA Protein Assay Kit (Thermo fisher, 23227) and equal amounts of proteins were loaded on SDS-PAGE gels. Immunoblotting analysis was performed as described previously 32 (link). The following primary antibodies were used with indicated dilution: Kindlin-2 (Proteintech, 11453-1-AP, 1:1000), DDX3X (Proteintech, 11115-1-AP, 1:1000), c-Myc (Cell signaling, 5605S, 1:1000), GAPDH (ABclonal, AC035, 1:5000), GFP (Transgen, HT801, 1:2000), Flag (Sigma, F1804, 1:3000), GST (Transgen, HT601, 1:2000) and MBP (Cell signaling, 2396S, 1:1000).

Western blot analysis was performed as described previously^{35 (link)}. The primary antibodies used in western blot were: Kindlin-2 (Proteintech, 1:1000 dilution), Fibronectin(FN) (Proteintech, 1:1000 dilution), Col1A1 (Boster, 1:400 dilution), α-SMA (Abcam, 1:100 dilution), Smad2/3 (Cell Signaling Technology, 1:1000 dilution), p-Smad2 (Cell Signaling Technology, 1:1000 dilution), p-Smad3 (Cell Signaling Technology, 1:1000 dilution), GAPDH (Abacm, 1:3000 dilution), β-actin (Abacm, 1:3000 dilution), and tubulin (Abcam, 1:3000 dilution). Detection was performed using a chemiluminescent substrate system (Bio-Rad). The gray values were analyzed with ImageJ software.