Specific hrp conjugated secondary antibodies

BC cells were lysed in ice-cold RIPA added with protease inhibitor cocktail. Lysates were subsequently sonicated and centrifuged at 12,000× g at +4 °C for 20 min. BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate protein concentration. Normalized protein lysates were separated by electrophoresis together with a prestained protein ladder (10–180 kDa, PageRuler, Thermo Fisher Scientific, Waltham, MA, USA) on 4–12% precast gels for SDS-PAGE (Invitrogen, Carlsbad, CA, USA). After gel running, proteins were transferred to Hybond-P pvdf membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 10% of BSA at room temperature, and subsequently incubated with primary antibodies (Table S2) overnight at +4 °C, and with specific HRP-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) for 1 h at room temperature. ECL Prime detection kit and Image Lab software (Bio-Rad, Hercules, CA, USA) were used for protein detection and quantification, respectively.

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).

Cells were lysed in ice-cold RIPA added with a protease inhibitor cocktail. Lysates were subsequently sonicated and centrifuged at 12,000× g at +4 °C for 20 min. The BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate the protein concentration. Normalized protein lysates were separated by electrophoresis together with a prestained protein ladder (10–180 kDa, PageRuler, Thermo Fisher Scientific) on 4–12% precast gels for SDS-PAGE (Invitrogen). After gel running, proteins were transferred to a Hybond-P pvdf membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 10% of BSA at room temperature and subsequently incubated with primary antibodies (Table S2) overnight at +4 °C, and with specific HRP-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) for 1 h at room temperature. The ECL Prime detection kit and Image Lab v5.2.1 software (Bio-Rad) were used for protein detection and quantification, respectively.