Anti p27 antibody

MCF-7 cells were seeded, cultured, and subjected to IC₅₀ of compound 6e (1.67 μM) for 48 h. Whole-cell protein lysates were prepared according to a standard protocol using RIPA buffer (Cell Signaling, Danvers, MA, USA). Total protein concentrations were determined in the supernatant using the Bradford method. Equal amounts (20 µg) of protein samples were mixed and boiled with SDS Loading buffer for 10 min, allowed to cool on ice and then loaded into SDS-polyacrylamide gel and separated by Cleaver electrophoresis unit (Cleaver, UK), transferred onto polyvinylidene fluoride (PVDF) membranes (BioRad, USA) for 30 min using a Semi-dry Electroblotter (Biorad, USA) at 2.5 A and 25 V for 30 min. Membranes were blocked with 5% non-fat dry milk in TBS-T and incubated overnight with the primary antibody: anti-p21, anti-p27 antibody, or anti-β-actin antibody (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), then incubated with secondary HRP-linked antibody (1:5000). Development was done using Pierce ECL2 chemiluminescent and chemifluorescent substrate (Perkin Elmer, Waltham, MA, USA). The chemiluminescent signals were captured using a CCD camera-based imager (Chemi Doc imager, Biorad, USA), and the bands’ intensities were then measured relative to actin by ImageLab (Biorad).

Total protein was extracted with RIPA buffer and quantified with bicinchoninic acid (BCA) protein quantitative assay (Thermo, USA). The samples were separated by 10% SDS-PAGE and transferred into PVDF membranes (Merck Millipore, USA). The membranes were blocked in 3% Bovine Serum Albumin (BSA) with PBS. Then, the membranes were incubated with corresponding antibodies: anti-ADSL antibody (Abcam, United Kingdom), anti-β-actin antibody (Cell Signaling Technology, USA), anti-Rb antibody (Cell Signaling Technology), anti-p21 antibody (Cell Signaling Technology), anti-CDK4 antibody (Cell Signaling Technology), anti-CDC2 antibody (Cell Signaling Technology), anti-Bcl2 antibody (Abcam), anti-Bax antibody (Abcam), anti-p27 antibody (Cell Signaling Technology), anti-Bid antibody (Cell Signaling Technology), anti-Bim antibody (Cell Signaling Technology). Appropriate second antibodies were applied in the next incubation. Lastly, the enhanced chemiluminescence (ECL) detection system (ImageQuant LAS 500, USA) was used to detect the membranes following the manufacturer’s protocol.