Ab32445

Cells were washed with PBS and lysed with lysis buffer (Beyotime). Total protein levels were measured using a BCA protein assay kit (Pierce). Western blotting was performed as described previously [13 (link)]. Briefly, 20 μg of total protein was mixed with 2× loading buffer and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Amersham). The PVDF membranes were blocked with 5% fat-free milk dissolved in Tris-buffered saline and Tween 20 (TBST) buffer for 1 h and then incubated with the primary antibodies (anti-CIT, Abcam, ab110897; anti-CCND1, Abcam, ab16663; anti-pBad, Cell Signaling Technology (CST), #9664; anti-Bad, Abcam, ab32445; and anti-GAPDH, Santa Cruz Biotechnology, sc-32233) overnight at 4°C. After three washes with TBST buffer, the secondary antibody (Santa Cruz Biotechnology, sc-2005) was added, and immune activity was detected using an ECL-Plus kit (Amersham Biosciences).

Total protein from BC and adjacent normal tissues or from cell lines, were extracted, separated by polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane through the wet transfer method. Next, the membrane was blocked in 5% bovine serum albumin (BSA) for 1 h and then probed with primary rabbit anti-human antibodies against Cosmc (sc-271829, Santa Cruz, California, U.S.A.), Ki67 (ab92742, 1:5000), proliferating cell nuclear antigen (PCNA; ab152112, 1:1500), Bcl-2-associated X protein (Bax; ab32503, 1:5000), and Bad (ab32445, 1:3000) (Cambridge, MA, U.S.A.) at 4°C overnight. The next day, the membrane was washed with Tris-Buffered Saline Tween-20 (TBST) (5 min× 3 times), and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (ab205718, 1:20,000) for 1 h. After development, ImageJ 1.48u software (National Institutes of Health, Bethesda, MA, U.S.A.) was used for protein quantitative analysis.