Anti ha monoclonal antibody

The constructions of the GLUT4 carrying an HA epitope in the extracellular loop (HA-GLUT4) or EGFP-tagged-GLUT4 (GLUT4-EGFP) were generated as described previously [9 (link), 10 (link)]. To detect cell-surface GLUT4, cells were plated in 6-well slides followed by different interventions. After 24 h of transfection, the cells were serum-starved for 2 h and then either treated or untreated with 100 nM insulin (Solarbio, China) for 30 min. After fixation with 4% paraformaldehyde, the cells were blocked with 5% BSA for 30 min at room temperature and incubated with anti-HA monoclonal antibody (1:1000, Cell Signaling Technology, USA) overnight at 4 °C, then washed three times and surface-bound monoclonal antibody detected using Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:200, Abcam, USA). Images were collected using a fluorescence microscope (Leica, Germany) and the mean fluorescence intensity was analyzed using ImageJ/Fiji software (N.I.H., Bethesda, MD). For co-localization assay, podocytes were co-transfected with exogenously expressed RAB3B-mCherry and GLUT4-EGFP. After washing with PBS and fixation with 4% paraformaldehyde, the cells were incubated with DAPI, and observed with a fluorescence microscope. Pearson’s correlation coefficient between RAB3B and GLUT4 was computed using the colocalization Finder of the ImageJ/Fiji software (N.I.H., Bethesda, MD).

The test and negative control slides used here were prepared as described in Daenzer et al [7 (link)]. Specifically, test slides included 5 micron sections of fixed liver tissue derived from GALT-null rats that had been administered an scAAV9-HA.hGALT vector either 14 or 30 days prior to euthanasia. Negative control slides included 5-micron sections of fixed liver tissue from GALT-null rats that had been administered vehicle alone (phosphate-buffered saline, PBS). The individual rats whose samples are presented here include: Rat A (FKRC311.6), Rat B (FKRC297.2), Rat C (FKRC297.12), Rat D (FKRC297.10) and Rat E (FKRC297.9).
Immunohistochemistry (IHC) was performed using an anti-HA monoclonal antibody (Cell Signaling Technology #3724) as primary at 1:1000 dilution, and a peroxidase-coupled goat anti-rabbit IgG (H+L) (Vector Labs PI-1000-1) as secondary. Antibody binding was visualized as brown color by reaction of the coupled peroxidase with 3,3′-diaminobenzidine (DAB; Vector Labs, SK-4100), as recommended by the manufacturer. All slides were also treated with hematoxylin which nonspecifically stained all cells a light blue color. Mounted slides were scanned on a Hamamatsu Nanozoomer 2.0 HT whole slide scanner at 40X and viewed using the free Hamamatsu software (NDP viewer).

The Co‐IP assays were performed as reported earlier (Cho et al., 2016; Yoon et al., 2017). Briefly, fusion molecules were co‐expressed in rice Oc cell protoplasts. Afterwards, fusion proteins were extracted in IP buffer [75 mm NaCl, 50 mm Tris‐HCl (pH 7.5), 5 mm EDTA, 1% Triton X‐100, 1 mm dithiothreitol, 1 mm phenylmethanesulfonylfluoride, 2 mm NaF, 20 μm MG132, and an appropriate amount of Protease inhibitor cocktail (Roche)]. Expressed proteins were immuno‐precipitated with anti‐HA mouse monoclonal antibodies (12CA5; Roche, Mannheim, Germany, http://www.roche.com) conjugated with A and G agarose beads (Millipore, Billerica, MA, USA, http://www.emdmillipore.com). For protein detection, we used horseradish peroxidase (HRP)‐conjugated anti‐Myc monoclonal antibody (#2040; Cell Signaling) and anti‐HA monoclonal antibody (#2999; Cell Signaling).