Bs 2540r

Western blots were performed on samples of pectoral major muscle, as previously described (Frolova et al. 2009 (link)). In total, 50 mg of muscle were grinded with liquid nitrogen and then the powder was lysed in 1 ml lysis buffer, 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 5 mM “ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, and completeMini protease inhibitor cocktail (BC3640; Solarbio). The samples were placed on ice for 4 min and then centrifugated at 12,000 rpm for 15 min at 4 °C. In total, 350 μl of the supernatant were transferred to a 2-ml auto-sampler vial and were diluted by 3-fold of loading buffer. Protein concentration was quantified using the BCA Protein Assay Kit (PC0020; Solarbio). Supernatant was then separated using Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h at 140 V and transferred to PVDF for 2 h at 90 V. Nonspecific antibody bind in was blocked in 5% nonfat dry milk powder in 1 Tris-buffered saline for 1 h. Blots were incubated in 1% nonfat dry milk powder in Tris-buffered saline with 0.05% Tween 20 overnight at 4 °C with the following antibodies: rabbit anti-GLUT12 (bs-2540R; 1:1,000; Bioss), and mouse anti-β-actin (bsm-33036M; 1:1,000; Bioss). Blots were incubated with goat anti-rabbit (bs-0295G; 1:3,000; Bioss) for 1 h at room temperature. Blots were detected using protein detection reagents (AY0371; Solarbio).

Rabbit monoclonal recombinant antibody to GLUT‐1 (ab115730), rabbit polyclonal antibody to GLUT‐3 (ab15311) and horse radish peroxidase (HRP) conjugated secondary goat anti‐rabbit IgG  (H + L) (ab205718) were purchased from Abcam, Waltham, MA, USA. Rabbit polyclonal antibodies to GLUT‐8 (BS‐4241R) and to GLUT‐12 (BS‐2540R) were obtained from Bioss Antibodies, Woburn, MA, USA. IHC Select® HRP/DAB kit was purchased from Merck Millipore, Darmstadt, Germany.

Cell monolayers were harvested with trypsin/EDTA solution followed by centrifugation (2000g, 4^oC, 10 minutes). Cell pellets were washed with phosphate buffered saline (PBS) before fixation overnight in 4% formalin buffered saline (4^oC). The spheroids were washed in PBS and also fixed overnight. After fixation, the cells and spheroids were processed through to wax and sectioned (5µM) then mounted onto glass slides. For immunohistochemical detection of GLUT-12, sections were de-waxed and rehydrated. Endogenous peroxidase activity was inhibited with 3% H₂O₂ in Tris-buffered saline (TBS) (20 minutes) before antigen retrieval was carried out (10mM citric acid, 0.5% Tween-20 in TBS (TBS-T) (pH6.0) at 100^oC for 20 minutes). Sections were blocked with 5% bovine serum albumin (BSA) in TBS-T (20 minutes). Sections were then incubated in rabbit anti-GLUT12 polyclonal antibody (1:200), (bs-2540R, Bioss, Woburn, MA, USA) 13 or GLUT1 (1:100) (GT12-A, ADI, San Antonio, TX, USA) overnight at 4ºC or incubated in TBS-T as a negative control. Sections were then washed in TBS-T before 2^oAb treatment (anti-rabbit IgG-HRP (Dako, Carpinteria, USA) room temperature for 1 hour). The sections were again washed then peroxidase activity was detected using 3´-3´-diaminobenzidine tetrahydrochloride before counter-staining with Haematoxylin.