Fig. 1

CAT1 protein expression on CC81-GREMG cells and newly established bovine CAT1 stably transfected reporter cell clones. a CAT1 expression histogram of flow cytometric analysis. Four CAT1 stably transfected clones (SC1, SC2, SC4 and SC5) were fixed with 1% formaldehyde/PBS and permeabilized with 0.1% TritonX-100/PBS prior to staining with rabbit anti-CAT1 polyclonal antibody (Abcam, Cambridge, UK) and AlexaFluor 647 goat anti-rabbit antibody. Permeabilization allowed antibody binding to an intracellular region of CAT1 protein. CAT1 protein expression was measured with a BD Accuri C6sampler plus (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software Ver.10 (FlowJo, LLC, Ashland, OR, USA). b Geometric mean expression of CAT1 in A. Mean and standard deviation of three independent experiments. The asterisk (*) represents a p-value of 0.05. c Western blot analysis of CC81-GREMG and CAT1 stably transfected single clone SC1 cell with anti-CAT1 (upper panel) and anti-β-actin antibodies (bottom panel). CC81-GREMG and SC1 cells were lysed with lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1.0% Nonidet P-40) and mixed with 4x SDS-sample buffer. Ten micrograms of total protein were applied to 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. CAT1 protein was detected by rabbit anti-CAT1 polyclonal antibody, horseradish peroxidase-conjugated rat anti-rabbit IgG antibody (Amersham Biosciences, Piscataway, NJ, USA), and SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific)