irs1  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: REP-1 deficiency induces aberrant mitochondrial metabolic rewiring from glycolysis to lipid oxidation in CHM disease

doi: 10.1038/s41419-026-08592-6

Figure Lengend Snippet: A Heat map representing the expression of the 16 genes associated with Glycolysis in ARPE-19 WT and ARPE-19 shREP1 cells. B Bubble plot representing the functional annotation analysis of the downregulated genes in Arpe-19sh-REP1 compared to WT. Counts indicate the number of genes found in each process. Color indicates the scale for the −log10 ( p value). C Physical interactions, generated by GeneMANIA, highlight REP-1, RAB10 and GLUT4 binding. D Representative immunofluorescence image of GLUT4-GFP and its translocation to the plasma membrane in basal and insulin-stimulated ARPE-19 WT and ARPE-19 shREP1 . Cells were transfected with GFP-tagged GLUT4. After 24 h cells were stimulated with Insulin (1 μM; 30’), fixed and analyzed by confocal microscopy. Nuclei were counterstained with Dapi. Scale bar 10 μm. E Schematic comparison of glycolysis metabolism in WT and REP-1 deficiency condition. In WT, GLUT4 guarantees glucose uptake in cytoplasm. When REP-1 is ablated, GLUT4 fails to reach the membrane efficiently, leading to impaired glucose uptake. F Representative images of Western Blot analysis of insulin receptor (IR), insulin receptor substrate 1 (IRS1) and some members of its signal transduction network PI3K p110, PI3K p85α, pAKT (ser473), AKT and GSK3β. β-Actin was used as loading control. Graphs show the levels of IR, IRS1, PI3K p110, PI3K p85 α, GSK3β and pAKT (ser473)/AKT ratio ± SEM ( n = 3 experiments at least). Statistical test: unpaired t -test. G Representative western blot of time course of insulin-induced phosphorylation of AKT in ARPE-19 WT and ARPE-19 shREP1 cells. Cells were treated with 1 µM of insulin in parallel at various time points for 30 min. β-Actin was used as loading control. Graph shows the mean of pAKT(ser473)/AKT ratio ± SEM (n = 3 experiments at least). Statistical test: unpaired t -test. H Immunoblots and calculated levels of GLUT1 and GLUT4. Data are expressed as value ± SEM of GLUT1 and GLUT4 relative to β-Actin, used as loading control. ( n = 3 experiments at least). Statistical test: unpaired t -test I Immunofluorescence labeling images of GLUT1 (green) and DAPI (blue) in ARPE-19 WT and ARPE-19 shREP1 . Scale bar 10 µm. J Representative confocal images of 2-NBDG in ARPE-19 WT and ARPE-19 shREP1 for glucose uptake evaluation after insulin stimulation (1 µM). Nuclei were counterstained with HOECHST (blue). Scale bar 50 µm. Graph shows the single values of the normalized fluorescence intensity (IntDen/Area) of 2-NBDG-positive cells ± SEM ( n = 3 experiments at least). Statistical test: unpaired t -test.

Article Snippet: For Western blot analysis, the following primary antibodies were used: mouse anti-REP1 (1:500, Santa Cruz SC-23905), rabbit anti-β actin (1:1000, Cell Signaling 4967), rabbit anti- Insulin Receptor (1:1000 Abclonal A19067), rabbit anti-IRS1 (1:1000, Abclonal A0245), rabbit anti-PI3 Kinase p110 (1:1000 Abclonal A0982), rabbit anti- PI3 Kinase p85 (1:1000, Abclonal A11177), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti- GSK3β (1:1000, Abclonal A11731), rabbit anti- GLUT1 (1:1000 Novus NB 300-666), mouse anti- GLUT4 (1:500 Santa Cruz SC-53566), rabbit anti-Citrate synthase (1:1000, Invitrogen PA5-22126), mouse anti-Tomm20 (1:500, Santa Cruz SC-136211), rabbit anti-LC3 (1:1000, Novus NB100-2220), mouse anti-total OXPHOS cocktail (1:500 Abcam ab110413).

Techniques: Expressing, Functional Assay, Generated, Binding Assay, Immunofluorescence, Translocation Assay, Clinical Proteomics, Membrane, Transfection, Confocal Microscopy, Comparison, Western Blot, Transduction, Control, Phospho-proteomics, Labeling, Fluorescence

Journal: Cell Death & Disease

Article Title: REP-1 deficiency induces aberrant mitochondrial metabolic rewiring from glycolysis to lipid oxidation in CHM disease

doi: 10.1038/s41419-026-08592-6

Figure Lengend Snippet: A Representative confocal image of 2-NBDG in ARPE-19 WT and ARPE-19 shREP1 for glucose uptake evaluation after insulin (1 µM) and leptin (150 nM) stimulation. Nuclei were counterstained with HOECHST (blue). Scale bar 50 µm. The Graph on the right shows single values of the normalized fluorescence intensity (IntDen/Area) of 2-NBDG-positive cells ± SEM (n = 3 experiments at least). Statistical test: unpaired t -test. B Immunofluorescence labeling images of GLUT1 (green) and DAPI (blue) in ARPE-19 WT and ARPE-19 shREP1 and leptin-treated ARPE-19 shREP1 . Scale bar 20 µm. C Representative immunofluorescence image of GLUT4-GFP translocation to the plasma membrane in insulin and leptin-stimulated ARPE-19 WT and ARPE-19 shREP1 . Cells were transfected with GFP-tagged GLUT4. After 24 h cells were stimulated with Insulin (1 µM; 30’) or leptin (150 nM; 30’), fixed and analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Scale bar 10 µm. Live cell imaging of GLUT4-GFP (green) vesicles translocation from the cytoplasm to the plasma membrane in ARPE-19 WT and ARPE-19 shREP1 cells without insulin stimulation (T0) and with a progressive insulin stimulation (from T30” to T4,5’), ( D ) and without leptin (T0) and with a progressive leptin stimulation ( E ). White boxes are magnifications showing GLUT4 migration. Scale bar 1 µm. F Immunoblots and calculated levels (right) of insulin receptor (IR), insulin receptor substrate1 (IRS1) and some members of its signal transduction network PI3K p110, PI3K p85α, pAKT (ser473), AKT, GLUT1, and GSK3β evaluated in ARPE-19 WT and ARPE-19 shREP1 and Leptin stimulated- shREP1 . Data are expressed as values of IR, IRS1, PI3K p110, PI3K p85, pAKT (ser473)/ AKT ratio, GLUT1, and GSK3β ± SEM ( n = 3 experiments at least). Statistical test: unpaired t -test.

Article Snippet: For Western blot analysis, the following primary antibodies were used: mouse anti-REP1 (1:500, Santa Cruz SC-23905), rabbit anti-β actin (1:1000, Cell Signaling 4967), rabbit anti- Insulin Receptor (1:1000 Abclonal A19067), rabbit anti-IRS1 (1:1000, Abclonal A0245), rabbit anti-PI3 Kinase p110 (1:1000 Abclonal A0982), rabbit anti- PI3 Kinase p85 (1:1000, Abclonal A11177), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti- GSK3β (1:1000, Abclonal A11731), rabbit anti- GLUT1 (1:1000 Novus NB 300-666), mouse anti- GLUT4 (1:500 Santa Cruz SC-53566), rabbit anti-Citrate synthase (1:1000, Invitrogen PA5-22126), mouse anti-Tomm20 (1:500, Santa Cruz SC-136211), rabbit anti-LC3 (1:1000, Novus NB100-2220), mouse anti-total OXPHOS cocktail (1:500 Abcam ab110413).

Techniques: Fluorescence, Immunofluorescence, Labeling, Translocation Assay, Clinical Proteomics, Membrane, Transfection, Confocal Microscopy, Live Cell Imaging, Migration, Western Blot, Transduction

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioinspired cardiac-targeted metal-organic framework nanozyme for modulating inflammatory responses in heart failure with preserved ejection fraction

doi: 10.3389/fbioe.2026.1744643

Figure Lengend Snippet: NanoAM regulates the SOCS3-IRS1-AKT2 axis to improve insulin resistance. (A,B) WB analysis of insulin resistance-related proteins in heart tissue (n = 6). (C) The Socs3 mRNA expression levels in heart tissues were determined by qPCR. (D) Immunofluorescence staining of GLUT4 in cardiac tissues. Scale bar, 10 µm. (E) Fluorescence intensity quantification of GLUT4 was shown. (F–I) The Inflammatory factors (IL-6,TNF-α,IL-1β,CRP) mRNA expression levels in heart tissues were determined by qPCR. (J,K) Serum levels of IL-6 and hs-CRP measured by ELISA. Data are showed as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s post hoc test; *p < 0.05, ***p < 0.001.

Article Snippet: Antibodies against IRS1-Ser307 (Proteintech, 85238-1-RR, dilution 1:5000), P-AKT1-T308+AKT2-T309+AKT3-T305 (ABclonal, AP1266, dilution 1:800), SOCS3 (Proteintech, 66797-1-Ig, dilution 1:20,000), and GAPDH (Servicebio, GB15002-100, dilution 1:8000) were used.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay