recombinant human glut2 protein  (Novus Biologicals)

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Evidence that hindbrain astrocytes in the rat detect low glucose with a glucose transporter 2-phospholipase C-calcium release mechanism

doi: 10.1152/ajpregu.00133.2019

Figure Lengend Snippet: Representative screenshots of identified astrocytes (color-coded encircled) and neurons (green arrows) adjacent to plots of the calcium-induced fluorescence signals over time evoked in these identified astrocytes by exposure to either ATP (viability test) and a glucoprivic [low glucose/2-deoxyglucose (LG/2DG)] challenge. Magnitudes of response to these challenges are reflected in the percent change in fluorescence of each individual astrocyte. Row 1 control conditions: A: screenshot of identified (double-labeled) astrocytes. B: adjacent trace shows the astrocytic responses to short exposure to ATP. C: the subsequent trace is representative of the robust astrocytic responses (color coded to the astrocytes encircled in A) to glucoprivic challenge under control conditions. Row 2 quercetin [glucose type 2 transporter (GLUT2) block] pretreatment: D: screenshot of identified astrocytes. E: adjacent trace are the astrocytic responses to short exposure to ATP. F: the subsequent trace is representative of diminished astrocytic responses (color coded to the astrocytes encircled in D) to glucoprivic challenge following pretreatment of slice with GLUT2 antagonist, quercetin. G: followed by another challenge with ATP to verify that the astrocytes were still viable after the GLUT2 blockade. Results following pretreatment with 2APB, U73122, and dantrolene are qualitatively similar (not shown here), and this is reflected in the summary data in Fig. 3. Row 3 fasentin (GLUT1/4 block) pretreatment: H: screenshot of identified astrocytes. I: adjacent trace showing astrocytic response to short exposure to ATP. J: astrocytes (color coded to cells encircled in H) response to LG/2DG challenge is not different from control. Row 4 U73122 (PLC block) pretreatment: K: screenshot of identified astrocytes. L: adjacent trace shows astrocytic response to short ATP exposure. M: U73122 blocks the effect of glucoprivic challenge on the identified astrocytes (color coded in K). Note that the ATP effect is mediated through a P2Y receptor that is also dependent on phospholipase C (PLC), so those effects are also blocked by U73122 (15) (null trace not shown).

Article Snippet: Five micrograms each of recombinant human Gαq protein (“GNAQ,” Abcam cat. no. ab132889) and recombinant human GLUT2 protein (Novus Biologicals cat. no. H00006514-P01) proteins were incubated at room temperature for 30 min. Five micrograms of IgG or anti-GNAQ antibody (rabbit polyclonal; Abcam cat. no. ab75825) were added and samples were incubated overnight at 4 ◦ C with end-over-end rotation.

Techniques: Fluorescence, Control, Labeling, Blocking Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Evidence that hindbrain astrocytes in the rat detect low glucose with a glucose transporter 2-phospholipase C-calcium release mechanism

doi: 10.1152/ajpregu.00133.2019

Figure Lengend Snippet: Averaged magnitude of changes in fluorescence due to intracellular calcium fluxes in hindbrain astrocytes in response to glucoprivic challenge after specific pretreatment conditions (number of astrocytes studied per each group is noted in parentheses). Exposure of astrocytes in hindbrain slices to the various pretreatment conditions produced significant differences in response to subsequent glucoprivic challenge. The “control” low glucose/2-deoxyglucose (LG/2DG) challenge yielded robust responses in viable astrocytes. In contrast, pretreatment of hindbrain slices with the selective glucose type 2 transporter (GLUT2) transporter blocker (quercetin) produced a nearly complete block of the LG/2DG effect, whereas the other transport antagonists phlorizin (SGLT) and fasentin (GLUT1/4) were not effective. Pretreatment with the phospholipase C (PLC) antagonist (U73122) completely blocked the subsequent effects of the LG/2DG challenge, whereas the inactive enantiomer (U73343) was without effect. Additionally, 2APB (an IP3 antagonist) also blocked the LG/2DG effect as did the endoplasmic reticulum (ER) ryanodine receptor antagonist, dantrolene. Low calcium Krebs did not eliminate the astrocyte response to LG/2DG. An overall one-way ANOVA yielded F8,441 = 33.11; P < 0.0001; Dunnett’s posttests: *P < 0.05.

Article Snippet: Five micrograms each of recombinant human Gαq protein (“GNAQ,” Abcam cat. no. ab132889) and recombinant human GLUT2 protein (Novus Biologicals cat. no. H00006514-P01) proteins were incubated at room temperature for 30 min. Five micrograms of IgG or anti-GNAQ antibody (rabbit polyclonal; Abcam cat. no. ab75825) were added and samples were incubated overnight at 4 ◦ C with end-over-end rotation.

Techniques: Fluorescence, Produced, Control, Blocking Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Evidence that hindbrain astrocytes in the rat detect low glucose with a glucose transporter 2-phospholipase C-calcium release mechanism

doi: 10.1152/ajpregu.00133.2019

Figure Lengend Snippet: Detection of a physical interaction between glucose type 2 transporter (GLUT2) and recombinant human Gαq protein (GNAQ). Recombinant GLUT2 and GNAQ were incubated at room temperature for 30 m followed by overnight immunoprecipitation (IP) with antibodies against either normal mouse serum (IgG) or GNAQ. Immunoprecipitated proteins were separated by SDS-PAGE, detected by immunoblotting (IB) with an antibody targeting GLUT2. The experiment was repeated on three separate occasions. The red arrow indicates detection of GLUT2 after immunoprecipitation using the GNAQ antibody.

Article Snippet: Five micrograms each of recombinant human Gαq protein (“GNAQ,” Abcam cat. no. ab132889) and recombinant human GLUT2 protein (Novus Biologicals cat. no. H00006514-P01) proteins were incubated at room temperature for 30 min. Five micrograms of IgG or anti-GNAQ antibody (rabbit polyclonal; Abcam cat. no. ab75825) were added and samples were incubated overnight at 4 ◦ C with end-over-end rotation.

Techniques: Recombinant, Incubation, Immunoprecipitation, SDS Page, Western Blot