Sc 21790

Cells were harvested at the logarithmic growth phase after overexpression treatment. The obtained cells were lysed in RIPA buffer supplemented with PMSF (RIPA: PMSF ratio of 99:1, Solarbio, Beijing, China), and total protein was quantified with the bicinchoninic acid (BCA) method (Biorad, China). The extracted proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto nitrocellulose membranes, blocked with non-fat milk (BD, USA) for 1 h at room temperature, and then incubated with primary antibodies against DDR1 (Catalog #sc-21790, Abcam), COL4A1 (eBioscience, Catalog# ab86042, Abcam), MMP-2 (Catalog # ab97779, Abcam), SLUG (Catalog #ab97779, Abcam), ZEB1 (Catalog #ab203829, Abcam), and β-actin polyclonal antibody (Catalog # ab179467, Abcam). β-actin was used as a control. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies rabbit anti-Mouse IgG H&L (1:5000, ab6728; 1:5000; Abcam) or donkey anti-rabbit IgG H&L (1:5000, ab6802, Abcam, USA). Using enhanced chemiluminescence (ECL) chromogenic substrate (Thermo Fisher), the antibody-reactive protein bands were visualized by the SuperSignal™ West Dura system (Pierce Biotechnology, Inc., Rockford, IL, USA), and UVITech imager (UVITech, Inc., Cambridge, UK) was used to photograph the membrane.

Immunohistochemical analysis was conducted on 30 BC tumor and adjacent non-tumorous tissues. Paraformaldehyde-fixed paraffin sections with a thickness of 6 µm were deparaffinized with xylene, and incubated for 15 min with 0.3% hydrogen peroxide to eliminate endogenous peroxidase activity. The sections were then treated with a blocking solution containing sheep serum for 50 min to block non-specific protein binding. After washing, the slides were stained with primary antibodies against DDR1 (Catalog #sc-21790, 1:200, Abcam), COL4A1 (eBioscience, Catalog# ab86042, 1:50, Abcam), and MMP-2 (Catalog # ab97779, 1:300, Abcam) overnight at 4°C. Cells were then incubated with rabbit anti-Mouse IgG H&L (1:5000, ab6728; 1:5,000; Abcam) or donkey anti-rabbit IgG H&L (1:5000, ab6802, Abcam, USA) conjugated with horseradish peroxidase at 37°C for 30 min. Finally, the sections were stained with 3′-diaminobenzidine and subsequently observed and photographed under an inverted microscope (IX71; Olympus Corporation). The integrated optical density (IOD) of the immunohistochemically stained samples from representative fields in each pathological section were calculated using Image-Pro Plus 6 software. The results were assessed semi-quantitatively.