mouse monoclonal antibodies  (Cell Signaling Technology Inc)

Journal: Biomolecules

Article Title: Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling.

doi: 10.3390/biom14101316

Figure Lengend Snippet: Figure 5. Effect of gintonin on GLUT4 expression in total lysates and plasma membrane fractions of C2C12 myotubes. (A) GLUT4 expression in total lysates. (B) GLUT4 expression in the plasma membrane fraction. C2C12 myotubes were treated with gintonin (GT, 10 µg/mL) for 120 min or insulin (INS, 100 nM) for 30 min. GLUT4 expression in total lysates and plasma membrane fraction of C2C12 myotubes was detected by immunoblotting. β-actin and Na+/K+ ATPase were also detected as loading controls. All data are shown as the mean ± SEM (n = 4); ** p < 0.01; *** p < 0.001 vs. untreated control cells (Con). Original western blot images can be found in Supplementary File S1.

Article Snippet: The cell membrane fraction and total cell lysate were also used to measure GLUT4 expression by immunoblotting using a mouse anti-GLUT4 monoclonal antibody (Santa Cruz Biotechnology) and a goat anti-mouse IgG antibody conjugated to HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot, Control

Journal: Biomolecules

Article Title: Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling.

doi: 10.3390/biom14101316

Figure Lengend Snippet: Figure 6. Possible signaling pathways of gintonin (GT)-induced glucose uptake in C2C12 myotubes. Gintonin induces transient increases in intracellular calcium concentrations and ERK activation via LPA receptor (LPAR) activation. These may lead to increases in the expression and translocation of GLUT4, subsequently increasing glucose uptake. PLC, phospholipase C; ERK, extracellular signal-regulated kinase; GLUT4, glucose transporter type 4.

Article Snippet: The cell membrane fraction and total cell lysate were also used to measure GLUT4 expression by immunoblotting using a mouse anti-GLUT4 monoclonal antibody (Santa Cruz Biotechnology) and a goat anti-mouse IgG antibody conjugated to HRP (Santa Cruz Biotechnology).

Techniques: Protein-Protein interactions, Activation Assay, Expressing, Translocation Assay

Journal: bioRxiv

Article Title: WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling

doi: 10.1101/2024.06.09.598125

Figure Lengend Snippet: A) Model shows downstream effects of insulin and the impact of WNKs on insulin signaling. B) Graph representing quantification of in vivo radioactive 2-deoxyglucose uptake per mg hippocampal weight in mice treated with vehicle or WNK463 (PO; 6 mg/kg); n=4. C) Graph representing enhanced radioactive 2-deoxyglucose uptake in hippocampal slice culture from C57BL/6J whole brains treated with WNK63 (1 µM); n=4. D) Graph representing enhanced radioactive 2-deoxyglucose uptake in crude synaptosome from C57BL/6J whole brains treated with WNK63 (1 µM); n=3. E) Graph shows in vitro radioactive 2-deoxyglucose uptake in SH-SY5Y cells treated with WNK463 (1 µM), insulin (10 nM); n=7. F) Graph representing enhanced radioactive in vitro 2-deoxyglucose uptake in differentiated SH-SY5Y cells treated with insulin (10 nM) ± WNK463 (1 µM) ± indinavir (10 nM); n=5. G) Representative Western blot shows surface and total GLUT4 protein fraction from mice hippocampal slices treated with insulin (10 nM) and/or WNK463 (1 µM). H) Corresponding quantification of ‘G’ shows enhanced surface GLUT4 (measured as a fraction of total GLUT4) with WNK463 ± insulin treatment; n=4. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t -test or one-way ANOVA. *p<0.05, **p<0.005 and *** p<0.0005. Graphics created with BioRender.com.

Article Snippet: WNK463 (Selleck Chemicals, S8358), anti-Vinculin antibody (Sigma Aldrich, V9131), anti-pOSR1/pSPAK antibody (EMD Millipore, 07-2273), anti-OSR1 polyclonal antibody (Cell Signaling, 3729S), anti-OSR1 monoclonal antibody (VWR, 10624-616), anti-WNK1 antibody (Cell Signaling, 4979S), anti-pAKT S473 antibody (Cell Signaling Technology, 4060S), anti-pAKT T308 antibody (Cell Signaling Technology, 4056S), anti-AKT1 antibody (Cell Signaling Technology, 2920S), anti-GAPDH antibody (Cell signaling Technology, 97166L), anti-GLUT4 polyclonal antibody (ab33780, Abcam), anti-GLUT4 monoclonal antibody (MA5-17176), anti-AS160 antibody (Cell Signaling Technology, 2670S), anti-pAS160 S588 antibody (50-191-485, Fisher Scientific), anti-sortilin antibody (MABN1792, Sigma-Aldrich), anti-Flag antibody (Sigma-Aldrich, F1804).

Techniques: In Vivo, In Vitro, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling

doi: 10.1101/2024.06.09.598125

Figure Lengend Snippet: A) Representative Western blot shows surface and total GLUT4 protein fraction in differentiated SH-SY5Y cells treated with insulin (10 nM) and/or WNK463 (1 µM). B) Corresponding quantification of ‘A’ shows enhanced surface GLUT4 (measured as a fraction of total GLUT4) with WNK463 ± insulin treatment; n=3. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t -test. *p<0.05, **p<0.005 and *** p<0.0005.

Article Snippet: WNK463 (Selleck Chemicals, S8358), anti-Vinculin antibody (Sigma Aldrich, V9131), anti-pOSR1/pSPAK antibody (EMD Millipore, 07-2273), anti-OSR1 polyclonal antibody (Cell Signaling, 3729S), anti-OSR1 monoclonal antibody (VWR, 10624-616), anti-WNK1 antibody (Cell Signaling, 4979S), anti-pAKT S473 antibody (Cell Signaling Technology, 4060S), anti-pAKT T308 antibody (Cell Signaling Technology, 4056S), anti-AKT1 antibody (Cell Signaling Technology, 2920S), anti-GAPDH antibody (Cell signaling Technology, 97166L), anti-GLUT4 polyclonal antibody (ab33780, Abcam), anti-GLUT4 monoclonal antibody (MA5-17176), anti-AS160 antibody (Cell Signaling Technology, 2670S), anti-pAS160 S588 antibody (50-191-485, Fisher Scientific), anti-sortilin antibody (MABN1792, Sigma-Aldrich), anti-Flag antibody (Sigma-Aldrich, F1804).

Techniques: Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling

doi: 10.1101/2024.06.09.598125

Figure Lengend Snippet: A) Model shows balanced regulation of sortilin and AS160 by the WNK/OSR1/SPAK pathway to regulate GLUT4 trafficking. B) Representative Western Blot shows co-immunoprecipitation of endogenous OSR1 with AS160 from whole brain C57BL/6J mouse lysates; n=3. C) Representative Western blot shows co-immunoprecipitation of AS160 with OSR1 upon treatment with WNK63 (1 µM) and/or insulin (10 nM) in differentiated SH-SY5Y cells. D) Corresponding quantification of ‘ C ’ shows decreased association of AS160 and OSR1 in cells treated with insulin (10 nM) + WNK463 (1 µM) compared to insulin alone; n=6. E) Representative Western blot shows pAS160, AS160 and GAPDH in differentiated SH-SY5Y cells treated with insulin (10 nM) ± WNK463 (1 µM). F) Corresponding quantification of ‘E’ shows increased pAS160 in cells treated with insulin (10 nM) or WNK463 (1 µM) compared to DMSO; n=4. G) R-x-F-x-V-containing blocking peptide (BP: WNK1 1253-1265; NH + -SAGRRFIVSPVPE-COO - ; 100 µM) decreases interaction of overexpressed OSR1/SPAK CCT bait protein fragment (aa 50-545) with myc-AS160 protein fragment (aa 193-446) in vitro ; n=3. H) Corresponding quantification of ‘G’. I) Bright-field (left) or confocal images shows co-localization of endogenous OSR1 with sortilin in differentiated SH-SY5Y cells. OSR1 (red), sortilin (green), merged (yellow), scale bar=10 µm; n=3. J) Representative endogenous co-immunoprecipitation of OSR1 with sortilin in differentiated SH-SY5Y cells; n=5. K) Yeast two-hybrid assay shows binding of the conserved C-terminus (CCT) of OSR1 with the C-terminus (C-t) of sortilin (3); binding of full-length and CCT of OSR1 to the C-terminus of WNK1 as positive controls (5,7). N-t: N-terminus. L) Representative Western blot shows co-immunoprecipitation of OSR1 and Flag-sortilin in HEK cells is diminished upon co-incubation with the blocking peptide (BP) SAGRRFIVSPVPE; n=3. M) Corresponding graphical representation for ‘L’; n=8. Data are represented as Mean±SE; analyzed by unpaired two-tailed Student’s t -test or one-way ANOVA. *p<0.05, **p<0.005 and *** p<0.0005. Graphics created with BioRender.com.

Article Snippet: WNK463 (Selleck Chemicals, S8358), anti-Vinculin antibody (Sigma Aldrich, V9131), anti-pOSR1/pSPAK antibody (EMD Millipore, 07-2273), anti-OSR1 polyclonal antibody (Cell Signaling, 3729S), anti-OSR1 monoclonal antibody (VWR, 10624-616), anti-WNK1 antibody (Cell Signaling, 4979S), anti-pAKT S473 antibody (Cell Signaling Technology, 4060S), anti-pAKT T308 antibody (Cell Signaling Technology, 4056S), anti-AKT1 antibody (Cell Signaling Technology, 2920S), anti-GAPDH antibody (Cell signaling Technology, 97166L), anti-GLUT4 polyclonal antibody (ab33780, Abcam), anti-GLUT4 monoclonal antibody (MA5-17176), anti-AS160 antibody (Cell Signaling Technology, 2670S), anti-pAS160 S588 antibody (50-191-485, Fisher Scientific), anti-sortilin antibody (MABN1792, Sigma-Aldrich), anti-Flag antibody (Sigma-Aldrich, F1804).

Techniques: Western Blot, Immunoprecipitation, Blocking Assay, In Vitro, Y2H Assay, Binding Assay, Incubation, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: Genetic deletion of zinc transporter ZnT 3 induces progressive cognitive deficits in mice by impairing dendritic spine plasticity and glucose metabolism

doi: 10.3389/fnmol.2024.1375925

Figure Lengend Snippet: ZnT 3 deletion is associated with a significant impairment of insulin signaling in the hippocampus. (A) Body mass analysis of ZnT 3 - / - and WT mice. *, ### Denote significant difference between groups indicated by the connector lines, p < 0.05, p < 0.001, two-way ANOVA with LSD post-hoc test. (B) Brain weight analysis of ZnT 3 - / - and WT mice. Two-way ANOVA with LSD post-hoc test. (C, D) The blood glucose levels in male (C) and female (D) mice (ZnT 3 - / - ; and WT) after fasting; n = 6 for both ZnT 3 - / - and n = 5 for both WT groups. Two-way ANOVA with LSD post-hoc test. (E, F) mRNA expression levels of Slc2a1 (GLUT1), Slc2a3 (GLUT3), and Slc2a4 (GLUT4) in cortex (E) ; and Slc2a1 (GLUT1), Slc2a3 (GLUT3), and Slc2a4 (GLUT4) and Insr (INSR) in hippocampus (F) . Expression levels are normalized to housekeeping gene Gapdh . * Denotes significant difference between groups indicated by the connector lines, p < 0.05, Mann-Whitney test. (G, H) Immunofluorescence staining of hippocampal sections of 9-months old mice. (G) : GLUT3-(red), DAPI-(blue) and GFAP (green). (H) : GLUT4-(green), DAPI-(blue) and NeuN (red). Scale bar, 50 μm; Zoom Scale bar, 5 μm. (I, J) Statistical analysis of images as those shown in (G) and (H) . * Denotes significant difference between groups indicated by the connector lines, p < 0.05, Mann-Whitney test.

Article Snippet: The following primary antibodies were used: rabbit anti-GLUT3 ployclonal antibody (20403-1-AP, Proteintech, USA; 1:2,000); mouse anti-GLUT4 monoclonal antibody (66846-1-lg, Proteintech, USA; 1:2,000); rabbit anti-GSK-3β monoclonal antibody (D5C5Z, Cell Signaling, USA; 1:1,000); and anti-GAPDH recombinant rabbit monoclonal antibody (SA30-01, HUABIO, China; 1:5,000).

Techniques: Expressing, MANN-WHITNEY, Immunofluorescence, Staining