Anti mct1 antibody

MCDB-131 cell culture medium was obtained from Gibco (Paisley, Scotland, UK). Other cell media, penicillin, streptomycin and amphotericin B, and trypsin were purchased from BioWhittaker (Verviers, Belgium). Fetal bovine serum (FBS) was purchased from Biowest (Kansas, USA). 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) and BODIPY FL C₁₆ (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid) were supplied by Molecular Probes (Eugene, OR, USA). L-[¹⁴C(U)]-Glutamine was acquired from Perkin Elmer (Waltham, Massachusetts, USA). Lactate assay kit was from Abnova (Taoyuan City, Taiwan). Anti-MCT1 antibody was from Santa Cruz Biotechnology (Heidelberg, Germany), anti-HIF-1α antibody was from BD Biosciences (Franklin Lakes, NJ, USA) and anti-LC3B and anti-α-tubulin antibodies were from Cell Signaling Technology (Danvers, MA, USA). Plastic material for cell culture was from Nunc (Roskilde, Denmark). All other reagents not listed on this section, including sodium palmitate, were from Sigma-Aldrich (St. Louis, MO, USA).

WNT5A-expressing breast cancer cells or parental breast cancer cells treated with PFKP siRNA, rWNT5A, XAV939, U0126 or not were washed with ice-cold PBS and lysed in ice-cold phosphorylation lysis buffer (PLB). The protein estimation, SDS-PAGE and visualization procedures were performed as described in Prasad et al. [40 (link)]. The following primary antibodies were used: anti-WNT5A from R&D systems (MN, USA); anti-Hexokinase-II, anti-Puruvate Kinase; anti-PKFP, anti-non-phospho (Active) β-catenin, and anti-pERK1/2 antibodies from Cell Signaling Technology (MA, USA); anti-MCT1 antibody from Santa Cruz Biotechnology Inc. (TX, USA); and anti-β-actin antibody from Sigma-Aldrich (MO, USA). The secondary antibodies used were goat anti-mouse, goat anti-rabbit and rabbit anti-goat, which were procured from Dako (Glostrup, Denmark). Separated protein bands were visualized using Chemiluminescence HRP substrate (Millipore), and the membranes were imaged and analyzed using the Chemi Doc™ imaging system from Bio-Rad.