rabbit anti-vnut  (Thermo Fisher)

Journal: iScience

Article Title: A purinergic mechanism underlying metformin regulation of hyperglycemia.

doi: 10.1016/j.isci.2023.106898

Figure Lengend Snippet: Figure 4. Hyperglycemia and P2Y2R dictate metformin’s antihyperglycemic action (A) Effect of high glucose loading (1.5g glucose/kg, i.p.) on the relative levels of mouse blood ATP at the indicated times. WT mice were pretreated for 15 min with saline or metformin (50 mg/kg, i.v.) through tail vein injection before glucose loading. n = 6 mice in each group. *p < 0.05 compared with the saline group; #p < 0.05 compared with the glucose group. One-way analysis of variance, followed by unpaired two-tailed Student’s t test. (B–D) Glucose tolerance test (GTT, 1.5g glucose/kg, i.p.) comparing P2Y2R-KO versus WT mice, metformin treatment (50 mg/kg, i.v.) versus saline in WT or P2Y2R-KO mice on a standard chow diet. n = 6–10, *p < 0.05. Unpaired two-tailed Student’s t test. (E) Insulin tolerance test (ITT, 0.75 IU insulin/kg, i.p.) comparing vehicle- versus metformin-treated mice on a standard chow diet. n = 6–9. (F and G) ITT was comparing saline versus metformin treatment on WT or P2Y2R-KO mice fed with a high-fat diet for 16 weeks n = 6–10, *p < 0.05. Unpaired two-tailed Student’s t test. (H) Immunohistochemical staining of VNUT and P2Y2R in liver sections of representative mice fed with normal chow or high-fat diet for 16 weeks. Scale bar: 200 mm.

Article Snippet: Then the cells were incubated with a rabbit anti-human SLC17A9 (VNUT) antibody (Proteintech) at 1:100 dilution overnight at 4 C, followed by incubation with an anti-rabbit IgG F(ab) fragment conjugated with AF-488 (Cell Signaling) at 1:1000 dilution for 90 min at room temperature in darkness.

Techniques: Saline, Injection, Two Tailed Test, Immunohistochemical staining, Staining

Journal: ACS Chemical Neuroscience

Article Title: Increased Synaptic ATP Release and CD73-Mediated Formation of Extracellular Adenosine in the Control of Behavioral and Electrophysiological Modifications Caused by Chronic Stress

doi: 10.1021/acschemneuro.2c00810

Figure Lengend Snippet: Repeated restrain stress triggered an increased density of vesicular nucleotide transporters and of ecto-5′-nucleotidase (CD73) in hippocampal and frontocortical nerve terminals. Male adult Wistar rats (8–10 weeks old) were subject to a protocol of restraint stress (4 h/day) during 14 days, evaluated behaviorally during 3 additional days, and then sacrificed for preparation of hippocampal and frontocortical synaptosomes. The immunoreactivity of vesicular nucleotide transporters (vNUT) (A, C) and of CD73 (B, D) was increased in synaptosomes from the hippocampus (A, B) and from the frontal cerebral cortex (C, D) of rats subject to repeated restraint stress (red) compared to control (black, equivalent to the dashed line corresponding to 100%), with representative Western blots shown below each bar graph. Data are the mean SEM of n = 6 different rats: * p < 0.05 and ** p < 0.01 one-tailed Student’s t test vs 100% (control).

Article Snippet: Western blot analyses of hippocampal synaptosomes were performed as described previously., , Briefly, the levels of ecto-5′-nucleotidase (CD73) were assessed using a previously validated rabbit polyclonal anti-CD73 antibody (1:300, Sigma, SAB5701396), and the levels of vesicular nucleotide transporters (vNUT, SLC17A9, solute carrier family 17 member 9) were estimated using a rabbit polyclonal anti-VNUT antibody (1:500; Santa Cruz Biotechnology, sc-86312), validated by others, after labeling with a secondary goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (1:20,000) (GE Healthcare, Chicago, USA).

Techniques: Western Blot, One-tailed Test

Journal: Frontiers in Pharmacology

Article Title: Haploinsufficient TNAP Mice Display Decreased Extracellular ATP Levels and Expression of Pannexin-1 Channels

doi: 10.3389/fphar.2018.00170

Figure Lengend Snippet: Panx1 is downregulated in TNAP+/– hippocampus. Representative Western blot and quantification of the protein expression of (A) VNUT, (B) , connexin 32 (Cx32) and connexin 43 (Cx43) and (C) Panx1 ( n = 4 WT and n = 5 TNAP+/–). Data are normalized to the expression levels of GAPDH “housekeeping” gene. Data are given as means ± SEM, ∗ p < 0.05 and ∗∗ p < 0.01, using unpaired Student’s t -test.

Article Snippet: Incubation with antibodies was performed overnight at 4°C at the dilutions specified in parentheses: goat anti-Pannexin-1 antibody (catalog SC49695, Santa Cruz Biotechnology, 1:100), mouse anti-connexin 32 antibody (catalog 13-8200, Life Technologies, 1:100), mouse anti-connexin 43 antibody (catalog 13-8300, Life Technologies, 1:100), rabbit anti-VNUT (catalog PA5-63312, Thermo Fisher Scientific, 1:50), rabbit anti-VNUT (catalog ABN110; Millipore, Billerica, MA, United States 1:500) or rabbit anti-GAPDH (catalog G9545, Sigma-Aldrich, 1:40.000).

Techniques: Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Haploinsufficient TNAP Mice Display Decreased Extracellular ATP Levels and Expression of Pannexin-1 Channels

doi: 10.3389/fphar.2018.00170

Figure Lengend Snippet: Panx1 is downregulated in TNAP+/– hippocampus. Representative Western blot and quantification of the protein expression of (A) VNUT, (B) , connexin 32 (Cx32) and connexin 43 (Cx43) and (C) Panx1 ( n = 4 WT and n = 5 TNAP+/–). Data are normalized to the expression levels of GAPDH “housekeeping” gene. Data are given as means ± SEM, ∗ p < 0.05 and ∗∗ p < 0.01, using unpaired Student’s t -test.

Article Snippet: Incubation with antibodies was performed overnight at 4°C at the dilutions specified in parentheses: goat anti-Pannexin-1 antibody (catalog SC49695, Santa Cruz Biotechnology, 1:100), mouse anti-connexin 32 antibody (catalog 13-8200, Life Technologies, 1:100), mouse anti-connexin 43 antibody (catalog 13-8300, Life Technologies, 1:100), rabbit anti-VNUT (catalog PA5-63312, Thermo Fisher Scientific, 1:50), rabbit anti-VNUT (catalog ABN110; Millipore, Billerica, MA, United States 1:500) or rabbit anti-GAPDH (catalog G9545, Sigma-Aldrich, 1:40.000).

Techniques: Western Blot, Expressing