Protein Fractionation and Western Blotting

Proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene difuoride (PVDF) membranes (Millipore,Shanghai, China),. PVDF membranes were blocked with 5% nonfat milk at room temperature for 2 h, and incubated with antibodies at 4 °C overnight,as described previously [42 (link)]. Primary antibodies were used as follows: anti-CDK4 (1:1000, Bioward Technology, Nanjing, China), anti-CDK5 (1:1000, Boster, Wuhan, China), anti-CDK6 (1:500, Bioward Technology), anti-cyclin D1 (1:500, Bioward Technology), anti-p27 (1:1000, Bioward Technology), anti-Notch1 (1:1000, Cell Signaling Technology, Boston, MA, USA), anti-NICD (1:1000, Abcam, Cambridge, MA, USA), anti- RBP-Jĸ (1:1000, Abcam), anti-Hes1 (1:1000, Cell Signaling Technology), and anti-β-actin (1:1000, ZSGB-BIO, Beijing, China). The secondary antibody was goat anti-mouse IgG (1:10,000, ZSGB-BIO) or goat anti-rabbit IgG (1:10,000, ZSGB-BIO), and enhanced chemiluminescence (ECL) detection (Millipore, Temecula, CA, USA). The exposure was performed using the Flour Chem HD2 imaging analyzer from ProteinSimple company.