Western Blot Analysis of BC Proteins

Western blot analysis was executed as previously described [23 (link)]. BC tissues and cells were lysed in lysis buffer (Beyotime). The bands were assessed through EZ-ECL chemiluminescence (Biological Industries, Beit-Haemek, Israel) and visualized using an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). The primary antibodies were as follows: anti-cyclin D1 (ab16663, 1:1,000; Abcam, Cambridge, MA, USA), anti-Cell Cycle Dependent Kinase 4 (CDK4) (ab108357, 1:500; Abcam), anti-GAPDH (ab128915, 1:5,000; Abcam), and anti-SCUBE2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit (ab97051, 1:10,000; Abcam) or mouse (ab205719, 1:2,000; Abcam) immunoglobulin G (IgG) was used as the secondary antibody. GAPDH was regarded as a loading control.