Ab237526

The kit used in protein lysis was radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), which was added with protease inhibitors (Bioss, Beijing, China). The kit used in concentration examination of proteins was the BCA kit (Thermo Fisher Scientific, Inc.). Proteins of equal quantity (30 μg) were separated by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The samples were then mounted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Then, an overnight incubation was performed on 5% skimmed milk powder-blocked membranes (1 h) as well as primary antibodies. The used antibodies were PRR11 (ab237526, Abcam, UK) and GAPDH (ab9485, Abcam, UK). Following 3 washes (10 min/time) with PBST, the treated membranes were subjected to 1 h of secondary antibody incubation (ab6721, Abcam, UK, goat anti-rabbit IgG, horseradish-labeled). Following 3 washes with PBST (10 min/time), the membranes were scanned under an optical illuminator (GE, USA).

To extract protein content from HCC tissue samples, RIPA lysis buffer (Beyotime, #P0013B) supplemented with PMSF protease inhibitor (Beyotime, #ST506, 1:100) was used. SDS-PAGE (Beyotime #P0012A) was used to separate the protein samples, and nitrocellulose membranes (Beyotime, #FFN53) were then used to transfer them. TBST (Beyotime, #ST671) containing 5% skimmed milk was used to block the membranes for 2 hours, followed by overnight incubation with primary antibodies at 4°C. Western blot analysis was performed with the following primary antibodies: KIF18A (abcam, #ab72417, 1:1000), CENPE (proteintech, #28142-1-AP, 1:1000), ATAD5 (abcam, #ab72111, 1:1000), KIAA1841 (Themo Fisher, #PA5-71136, 1:500), CDCA2 (proteintech, #17701-1-AP, 1:1000), PRR11 (abcam, #ab237526, 1:1000), TMEM164 (abcam, #ab122510, 1:250), and GAPDH (proteintech, #10494-1-AP). An additional hour of secondary antibody (abcam, #ab205718; #ab6721) incubation was performed after the primary antibody incubation. Chemiluminescence imaging systems (clinx, #ChemiScope 6000 Touch) were used to detect protein bands using the ECL chromogenic kit (Thermo Fisher, # 32106).