human slc2a2  (Novus Biologicals)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Function of Organic Cation Transporter 2 in Pancreas

doi: 10.3389/fcell.2021.688885

Figure Lengend Snippet: This figure shows the expression of GLUT and OCT in the pancreas. In panel (A) a representative immunofluorescence analysis of GLUT2 (green) and OCT2 (red) expression in a mouse pancreatic islet is shown. GLUT2 is evident in the plasma membrane of islet cells. OCT2 seems to be expressed in intracellular compartments of some islet cells. No GLUT2/OCT2 co-localization is observed. Panel (B) shows that mouse pancreas expresses mRNA of GLUT1 and 2 and of OCT2, but not of OCT1 and 3. For OCT2 several bands are visible, but sequencing confirmed the identity of the lower band with OCT2. In rat pancreas (C) OCT expression became visible only after reamplification. However, this reamplification caused the appearance of an unspecific band for GLUT1. Sequencing confirmed the lower band to be specific for GLUT1. The INS-1 cells show the same transporter expression pattern as the rat pancreas (D) .

Article Snippet: The effect of transfection with human GLUT2 (hGLUT2, a gift from RESOLUTE Consortium & Giulio Superti-Furga, Addgene plasmid #132126 ) cloned into a pcDNA DEST47 vector) was studied in human embryonic cells (HEK) stably over-expressing the human OCT2 [hOCT2, for a detailed description of these cells see ].

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Function of Organic Cation Transporter 2 in Pancreas

doi: 10.3389/fcell.2021.688885

Figure Lengend Snippet: This figure shows the ASP + transport characteristics of INS-1 cells and the analysis of OCT2 and GLUT2 expression in dependence from glucose concentration. Panel (A) shows that TPA + inhibits concentration-dependently the ASP + uptake with an IC 50 of 519 μM. Inhibition of ASP + uptake by metformin is also shown (at least six replicates for concentration measured in three independent experiments). Panel (B) shows the effect of incubation with 14.8 mM glucose for 1, 24, or 48 h on the ASP + uptake in arbitrary units (a.u.) of INS-1 cells. Control experiments were performed with a glucose concentration of 9.8 mM. Osmolarity was adjusted in control experiments by adding mannose. High glucose significantly stimulated ASP + uptake after incubation for 24 and 48 h (ANOVA with Tukey’s post-test, nine replicates measured in four independent experiments). Panel (C) shows the evaluation of western blot analysis of OCT2 expression under incubation with high glucose. Only after 48 h incubation with 14.8 mM glucose a significant increase of OCT2 expression was observed (*, ANOVA with Tukey’s post-test, three independent experiments). The insert on the left shows an example of the western blot analysis. Panel (D) shows the immunofluorescence analysis of OCT2 (red) and GLUT2 (green) expression in INS-1 cells after 48 h incubation with normal (9.8 mM) or high (14.8 mM) glucose. The labeling of nuclei with 4′,6-diamidino-2-phenylindole (DAPI, blue) is also shown. In the upper right corners of the overlay picture a magnification of the field indicated by a box is shown.

Article Snippet: The effect of transfection with human GLUT2 (hGLUT2, a gift from RESOLUTE Consortium & Giulio Superti-Furga, Addgene plasmid #132126 ) cloned into a pcDNA DEST47 vector) was studied in human embryonic cells (HEK) stably over-expressing the human OCT2 [hOCT2, for a detailed description of these cells see ].

Techniques: Expressing, Concentration Assay, Inhibition, Incubation, Control, Western Blot, Immunofluorescence, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Expression and Function of Organic Cation Transporter 2 in Pancreas

doi: 10.3389/fcell.2021.688885

Figure Lengend Snippet: Transfection of HEK cells stably expressing hOCT2 with GLUT2: effects on the ASP + uptake. Panel (A) shows the uptake of 1 μM ASP + in HEK cells 72 h after transfection with GLUT2 or empty vector (EV). Overexpression of GLUT2 significantly inhibited ASP + uptake (*, unpaired t -test, 27 replicates measured in three independent experiments). Panel (B) shows the western blot analysis of GLUT2 72 h after transfection. α-Actinin was used as loading marker. This antibody against hGLUT2 is known to detect mature glycosylated hGLUT2 at 60-70 kDa, non-glycosylated hGLUT2 at 38–45 kDa, and a hGLUT2 fragment at around 35 kDa ( https://www.ptglab.com/products/SLC2A2-Antibody-20436-1-AP.htm ). All these bands are visible in this western blot analysis, especially in HEK cells transfected with hGLUT2, confirming that transfection with hGLUT2 was successful. Bands corresponding to GLUT2 were observed mainly in transfected cells.

Article Snippet: The effect of transfection with human GLUT2 (hGLUT2, a gift from RESOLUTE Consortium & Giulio Superti-Furga, Addgene plasmid #132126 ) cloned into a pcDNA DEST47 vector) was studied in human embryonic cells (HEK) stably over-expressing the human OCT2 [hOCT2, for a detailed description of these cells see ].

Techniques: Transfection, Stable Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot, Marker

Journal: Nature chemical biology

Article Title: Synthetic beta cells for fusion-mediated dynamic insulin secretion

doi: 10.1038/nchembio.2511

Figure Lengend Snippet: (a) Schematic of the biochemical processes inside the AβCs. GOx, glucose oxidase; CAT, catalase; square brackets denote concentration. (b) Top, ISVs stained with uranyl acetate and imaged by TEM. The ISVs were used to mimic the insulin granules inside natural beta cells (scale bar, 100 nm). Bottom, size-distribution histogram of the insulin-containing ISVs. (c) Top, cryo-SEM micrograph of the vesicles-in-vesicle superstructures (scale bar, 5 μm). Bottom, size distribution of vesicles-in-vesicle superstructures (ISVs@OLV). (d) Magnified fractured cryo-SEM micrograph of the vesicles-in-vesicle superstructures (scale bar, 200 nm). From the fracture in d, small liposomal vesicles can be clearly seen inside the large liposome. (e) CLSM micrographs verifying the encapsulation of glucose oxidase labeled with fluorescein isothiocyanate (GOx-FITC) and catalase labeled with rhodamine B (CAT-RB) inside the large liposomes (scale bar, 5 μm). (f) Western blotting results indicating the retention of immunoreactivity of GLUT2 in the superstructures. The full gel is shown in Supplementary Figure 9. (g) CLSM image showing the reconstitution of GLUT2 labeled with rhodamine B on the membranes of the larger liposomes (scale bar, 5 μm). (h) CLSM image showing insertion of the proton channel gramicidin A labeled with lysine-5-carboxyfluorescein into the outer membrane (scale bar, 5 μm).

Article Snippet: GLUT2 expression and purification The cDNA encoding mouse GLUT2 (Origene, GenBank {"type":"entrez-nucleotide","attrs":{"text":"NM_031197","term_id":"165377236","term_text":"NM_031197"}} NM_031197 ) was amplified and cloned into the BamHI and HindIII sites of pET-28a (Novagen; full plasmid sequence in Supplementary Note ).

Techniques: Concentration Assay, Staining, Labeling, Western Blot