Ab254324

MTEC1 cells were inoculated into 6-well culture plates. When the cell fusion degree reached 60%, the overexpression vector, siRNA, miRNA, mimic and negative control were transfected into the cells. After 48 h, cell proteins were extracted using RIPA buffer and protease inhibitor mixture and then separated by 10% SDS-PAGE and immunoblotted using various antibodies according to standard western blot procedures. Rabbit anti-IGFBP5 (ab254324; Abcam, Cambridge, UK), rabbit anti-p53 (5395S; Cell Signaling Technology, Danvers, USA), rabbit anti-p21 (2947S; Cell Signaling Technology), rabbit anti-Bax (5023P; Cell Signaling Technology), rabbit anti-Cyclin B1 (ab55184; Abcam), rabbit anti-Cyclin D1 (ab134175; Abcam) and rabbit anti-β-actin (ab179467; Abcam) of 1:1000 dilution were incubated with protein blots at 4°C overnight. Subsequently, the secondary antibody (goat anti-rabbit) (A0208; Beyotime) of 1:1000 dilution was use to incubate with the blots at 37°C for 1 h. Finally, chemiluminescence detection was carried out using the ECL Plus (Solarbio, Beijing, China).

Total protein samples of tissues and cultured cells were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Approximately 10 μg protein samples were separated on 12% SDS-PAGE and transferred onto PVDF membranes. After blocked with 5% skimmed milk, the membranes were incubated with primary antibodies against IGFBP5 (Abcam, ab254324. 1:500), Ki67 (Abcam, ab15580. 1:500), and GAPDH (Abcam, ab9485, 1:1000) overnight at 4 °C. On the following day, the membranes were exposed to horseradish peroxidase-conjugated secondary antibody for 2 h. The signals were detected using an enhanced chemiluminescence detection kit (ECL, Bio-Rad, Hercules, CA, USA).