Novex chemiluminescent substrate reagent kit

Protein lysates from cell and tissues were lysed and homogenized (tissues) in Mammalian Protein Extraction Reagent (M-PER) lysis buffer (Cat. no. 78501; Thermo Fisher Scientific) supplemented with cOmplete protease inhibitor tablets (Cat. no. 1183617000; MilliporeSigma). Approximately 50 μg of lysate were electrophoretically resolved on 4–20% Novex Tris-glycine gradient gels (Cat. no. XV04200PK20; Thermo Fisher Scientific). Proteins were then transferred to 0.2-μm PVDF membranes (Cat. no. 88520; Thermo Fisher Scientific) and incubated overnight at 4°C with gentle rocking in primary antisera diluted 1:1,000 in 5% bovine serum albumin (Cat. no. A30075; RPI Corp)/1xTBS-Tween (Cat. no. IBB-581X; Boston BioProducts). Membranes were washed three times in 1xTBS-Tween for 5 min each and then incubated in secondary antisera (1:2,000 dilution) for 1 h at room temperature. Membranes were then washed four times in 1× TBS-Tween for 15 min each before the addition of the Novex Chemiluminescent Substrate Reagent Kit (Cat. no. WP20005; Thermo Fisher Scientific). Membranes were exposed onto PerfectFilm audioradiography film (Cat. no. B581; GenHunter) and developed on a Typhoon Variable Mode Imager (Amersham Pharmacia). Western blot densitometry was performed using open-source ImageJ software.

TNBC cells or tissues were lysed by RIPA. BSA method was used for the determination of total proteins, and 15 μL protein sample was loaded in the lane at a protein concentration of 1 mg/mL. Then, SDS-PAGE (12% gel) was performed. After adding the transmembrane onto NC membranes, membranes were blocked with 5% fat-free milk in TBST at room temperature and subsequently incubated with the primary antibodies of CCNB2 and β-actin at room temperature for 1.5 h. Then, the NC membranes were incubated with HRP-conjugate secondary antibodies for 1 h at room temperature. Signals were detected using ECL kit (Novex™ Chemiluminescent Substrate Reagent Kit, Thermo Fisher).