Rcl1 15330 1 ap

Cells were lysed in RIPA buffer (Beyotime Biotechnology, China) containing 1X Protease and Phosphatase inhibitor (Beyotime Biotechnology, China). An equal amount of protein samples was separated by 8% SDS/PAGE and transferred to 0.25 mm polyvinylidene fluoride membranes (Millpore, Germany). The membranes were blocked with 5% non-fat milk for 1 h, then incubated with the individual antibody at 4 ℃ overnight: Tubulin (YFB6011, YIFAN BIOLOGICAL), Rcl1 (15330-1-AP; Proteintech), GAPDH (bs-0755R, Bioss). The membranes were then incubated with the second antibody (bs-40295G-HRP, Bioss) at 37 ℃ for 1 h. Finally, protein bands were visualized using Omni ECL reagent (EpiZyme, China), and the gray intensity was acquired by using Fiji (NCBI, USA).

HCC cells were seeded on a 35 × 35 mm confocal cell culture plate (Thermal, USA). After fixed using 4% paraformaldehyde, cultured cells were blocked with Immunol staining blocking buffer (Beyotime, China) with 0.3% Triton X-100 (Beyotine, China) in PBS for 30 min at RT. The samples were then incubated with primary antibody overnight at 4 ℃: Rcl1 (15330-1-AP; Proteintech), followed by the appropriate secondary fluorescently labeled antibodies (Invitrogen, USA) for 1 h at 37 ℃. Nuclei were counterstained with DAPI (Beyotime). Images were analyzed by a laser scanning confocal microscope (Olympus, FV300).