Protein Expression Analysis by Western Blot

Protein expression analysis was detected by Western blot analysis as previously described [29 (link), 40 (link)]. The membranes were incubated with primary antibodies, including anti-BMF (ab9655, 1:1000, Abcam), anti-Runx2 (ab76956, 1:1000, Abcam), anti-p16 (10883-1-AP, 1:1000, Proteintech), anti-p21 (10355-1-AP, 1:1000, Proteintech), and anti-GAPDH (sc-365062, 1:3000, Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated goat anti-rabbit (sc-2004, 1:5000, Santa Cruz Biotechnology) or goat anti-mouse (sc-2005, 1:5000, Santa Cruz Biotechnology) secondary antibodies at 37°C for 1 hour. The immunoreactive bands were visualized using the ECL Plus Western blot detection kit (Amersham Biosciences U.K. Ltd) and densitometric quantification of band intensity from three independent experiments was carried out with the Image-Pro Plus 6.0 software. The relative expression level of target protein was normalized to the intensity of the GAPDH band.