Ab192237

Cells were washed 3 times with cold, sterile phosphate-buffered saline (PBS), and then lysed by addition of 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Cell lysates were collected into tubes and centrifuged at 12 000 g for 15 minutes at 4°C. The supernatants and protein loading buffer (SDS-PAGE gel, CWBIO, Beijing, China) were mixed at a ratio of 4: 1 according to the instructions and then divided into 2 aliquots. One aliquot was fractionated by SDS-PAGE, and the other was stored at −80°C. All the western blotting procedures were performed according to the manuals. In this study, the primary antibodies used were as follows: a rabbit anti-human CDCA5 (ab192237, 1: 1000, Abcam, Cambridge, MA, USA), a rabbit anti-human CDK1 antibody (ab32094, 1: 1500, Abcam, Cambridge, MA, USA), a rabbit anti-human β-actin antibody (ab8227, 1: 1000; Abcam, Cambridge, MA, USA). An HRP-labeled goat anti-rabbit antibody (A0208, 1: 1000, Beyotime, China) was used as the second antibody. The band signals were detected using chemiluminescence (Millipore, MA, USA) by infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The target protein was normalized to β-actin through a comparison of the gray scale values, and the analysis was performed by Quantity One (version 4.6.2).