Bs 1155r

Cells were washed with PBS buffer and lysed with lysis buffer to extract total protein, which was quantified with a BCA Protein Assay Kit (BCA Protein Assay Kit, Thermo Fisher Scientific, USA). For western blot (WB) analysis, 30–40 μg protein samples were mixed with loading buffer. Cell lysates were then separated by SDS–PAGE electrophoresis and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% milk in TBST buffer for 1 h and then incubated with the primary antibody overnight at 4°C. The primary antibodies used here are as follows: mouse anti-RB, Cell Signaling Technology, 9,309 (1:2000), rabbit anti-EZH2, Cell Signaling Technology, 5,246 (1:1000), rabbit anti-TTK, Cell Signaling Technology, 3,255 (1:1000), rabbit anti-NUSAP1, Proteintech, 12,024-1-AP (1:1000), rabbit anti-UBE2C, Proteintech, 12,134-2-AP (1:1000), rabbit anti-GAPDH, Cell Signaling Technology, 2,118 (1:4000), rabbit anti-ASCL1, Bioss, bs-1155R (1:1500). After 3 washes with TBST buffer, specific HRP-conjugated secondary antibodies from Jackson ImmunoResearch were added, and immunoreactivity was detected with the ECL-Plus kit (Thermo Fisher Scientific, United States).