Gapdh monoclonal antibody

Cells were washed with twice pre-cold PBS and lysed in RIPA Lysis Buffer (Millipore, Bedford, MA, USA). Protein concentrations were quantified by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Protein lysates were resolved on a 10% SDS-polyacrylamide gel and electroblotted onto Immobilon-P PVDF membranes (Millipore). The membranes were blocked in TTBS containing 5% nonfat milk and incubated with diluted primary antibodies overnight at 4 °C. The membranes were then reacted with species-specific HRP-conjugated secondary antibodies (1:5000–10000 in TTBS), and the immunoreactive protein bands were detected by Amersham ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). The primary antibodies used included Brachyury monoclonal antibody (1:1000; Millipore), GAPDH monoclonal antibody (1:30000; Chemicon, Temecula, CA, USA), GFAP monoclonal antibody (1:2000; Millipore), NANOG polyclonal antibody (1:2000; Abcam, Cambridge, UK), NeuroD1 polyclonal antibody (1:2500; Millipore), OCT4 monoclonal antibody (1:2000; Cell Signaling Technology, Beverly, MA, USA), Pax6 monoclonal antibody (1:2000; Abnova, Taipei, Taiwan), and SOX2 polyclonal antibody (1:2000; Chemicon).

Total protein from MCF-7, MDA-MB-231, MDA-MB-468, SKBR-3 and SUM159 cell lysates were extracted by resuspending the cell pellets in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 7.4) and 1% Triton X-100). The protein concentration was measured using a BCA Protein Assay Kit (Cat# 23227, Thermo, USA). Approximately 30 μg of total protein per sample was separated by SDS-PAGE and then transferred onto nitrocellulose membranes. Western blot analyses were performed with polyclonal antibodies against GPD1 (Cat# sc-376219, Santa Cruz Biotechnology, USA), with a monoclonal GAPDH antibody as a control (Cat# G9545, Sigma, USA).