rabbit anti glut4 polyclonal ab  (Novus Biologicals)

Journal: Molecular metabolism

Article Title: Pancreatic beta-cell specific BAG3 knockout results in chronic hyperinsulinemia inducing insulin resistance.

doi: 10.1016/j.molmet.2023.101752

Figure Lengend Snippet: Figure 5: BAG3bKO mice displayed peripheral but not hepatic insulin resistance. (A) Venn diagram and volcano plot showing the number of proteins and DEPs detected in muscles of 64-weeks old control and BAG3bKO mice. Down-regulated and up-regulated DEPs identified by MS/MS analysis are highlighted in blue and red, respectively. (B) Significantly inhibited upstream predicted by IPA analysis. (C, D) Representative Western blot of muscle INSR, GLUT4 phospho-AKT/AKT, and phospho-ERK1/2/ERK1/2 expression in control and BAG3bKO mice. Relative protein expression data are presented as mean values SEM; n ¼ 7e8 for each group; *p < 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with primary antibodies: anti-BAG3 used 1:2000 (Novus Biologicals, Milan, Italy), anti-INSR used 1:1000, anti-AKT used 1:1000, anti-phospho-AKT used 1:1000, anti-ERK1/2 used 1:1000, antiphospho-ERK1/2 and anti-GAPDH used 1:2000, all supplied by Cell Signaling Technology (Danvers, MA, USA), anti-GLUT4 Rabbit polyclonal Ab used 1:1000 (Antibodies.com, Cat. #95872), anti p62/ SQSTM1 used 1:1000 (BD Bioscience, San Jose, CA, USA), anti-Insulin B (C-12) used 1:500 (Santa Cruz Biotechnology) and anti-b-actin used 1:40,000 (SigmaeAldrich, Milan, Italy).

Techniques: Muscles, Control, Tandem Mass Spectroscopy, Western Blot, Expressing

Journal: Brain Communications

Article Title: Differential regulation of insulin signalling by monomeric and oligomeric amyloid beta-peptide

doi: 10.1093/braincomms/fcac243

Figure Lengend Snippet: mAβ 1-40 induces GLUT4 membrane translocation. ( A ) Scheme of the intracellular effects of IR activation. Insulin binds the IR inducing its dimerization and activating the receptor to cause its autophosphorylation. This produces the binding and phosphorylation of the signalling adapter protein IRS. IRS activates several downstream pathways, but here we focus on PI3K/Akt system. The activation of PI3K leads to the phosphorylation of Akt, which produces the GLUT4 translocation to the membrane, allowing glucose uptake by cells. ( B, C ) mAβ 1-40 induces GLUT4 translocation. Cells were treated for 10 min with 100 nM insulin or 150 nM mAβ 1-40 , afterwards cells were fixed. Extracellular expressed GLUT4 was labelled with an anti-GLUT4 Ab and nuclei were stained with DAPI. ( B ) Representative images of cells used in ( B ). Bars represent 20 nm. ( C ) GLUT4 fluorescence intensity at 555 nm was quantified. Data are the mean ± SEM of 4 independent experiments. ** P < 0.01 compared with untreated controls by one-way ANOVA plus Student–Newman–Keuls as post hoc test. ( D ) Cells were treated as in ( B, C ). Glucose uptake was measured and expressed regarding untreated controls. Data are the mean ± SEM of five independent experiments. *** P < 0.01, * P < 0.05 compared with controls by one-way ANOVA plus Student–Newman–Keuls as post hoc test.

Article Snippet: This procedure was performed using 1:100 rabbit anti-GLUT4 Ab (NBP1-49533; Novus Biologicals).

Techniques: Membrane, Translocation Assay, Activation Assay, Binding Assay, Phospho-proteomics, Staining, Fluorescence

Journal: Brain Communications

Article Title: Differential regulation of insulin signalling by monomeric and oligomeric amyloid beta-peptide

doi: 10.1093/braincomms/fcac243

Figure Lengend Snippet: oAβ 1-40 blocks IR activation. ( A ) oAβ 1-40 binds to IR impairing its autophosphorylation. Human neuroblastoma cells were pre-treated with increasing concentrations of oAβ 1-40 for 30 min and then treated in the presence/absence of insulin (100 nM) for 10 min. The upper panel shows a representative WB with anti-p-IR, anti-p-Thr308-Akt and anti-GAPDH Abs. ( B ) Quantification of the p-IR bands and normalized by GAPDH obtained by WB as described in ( A ). Data are the mean ± SEM of 4–5 independent experiments. * P < 0.05 compared with untreated control cells by one-way ANOVA plus Student–Newman–Keuls as post hoc test. ( C ) Quantification of the p-Thr308-Akt bands and normalized by GAPDH obtained by WB as described in ( A ). Data are the mean ± SEM of 4–5 independent experiments. * P < 0.05 regarding untreated control cells by one-way ANOVA plus Student–Newman–Keuls as post hoc test. ( D ) oAβ 1-40 do not induce the translocation of GLUT4. Representative images of cells treated with 100 nM insulin and 150 nM oAβ 1-40 for 10 and 30 min, respectively. Extracellular GLUT4 was labelled with an anti-GLUT4 Ab and nuclei are stained with DAPI. Bars represent 20 nm. ( E ) oAβ 1-40 does not induce glucose uptake. Cells were treated as in ( A ) and glucose uptake was measured as indicated in the M&M section and expressed regarding untreated controls. Data are the mean ± SEM from five independent experiments. ** P < 0.01, n.s., non-significant compared with untreated cells by one-way ANOVA plus Student–Newman–Keuls as post hoc test.

Article Snippet: This procedure was performed using 1:100 rabbit anti-GLUT4 Ab (NBP1-49533; Novus Biologicals).

Techniques: Activation Assay, Control, Translocation Assay, Staining