Journal: Cell Discovery

Article Title: VAPB-mediated ER-targeting stabilizes IRS-1 signalosomes to regulate insulin/IGF signaling

doi: 10.1038/s41421-023-00576-6

Figure Lengend Snippet: a The gel lane was subjected to trypsin digestion followed by MALDI-TOF analysis, and VAPB was identified as a potential interacting partner of IRS-1. Peptides identified by MS analysis of FLAG-IRS-1 immunoprecipitates were listed. b C2C12 myoblasts, treated with or without 100 ng/mL IGF-1 for 2.5 min after serum starvation for 16 h, were subjected to immunoprecipitation with IRS-1 antibodies. Coimmunoprecipitated IRS-1 and VAPB were detected by Western blot analysis. The relative IRS-1 levels co-precipitated by VAPB were quantified. * P < 0.05. c FLAG-tagged IRS-1 or 9YA mutant was co-transfected with HA-VAPB into 293 T cells for co-IP analysis. d GST or GST-VAPB fusion protein was incubated with purified FLAG-tagged IRS-1 for direct Pull-down assay. e Schematic diagram of VAPB and its mutants. f FLAG-tagged IRS-1 or mutant constructs as shown in ( e ) were co-transfected with GFP-IRS-1 into 293 T cells for co-IP assays. g FLAG-tagged IRS-1 was co-transfected with HA-VAPB or VAPB KMDD mutant into 293 T cells for co-IP analysis.

Article Snippet: C2C12 cells were serum-starved for 12 h in DMEM, and then treated with 100 ng/mL IGF-1 (Sino biological, 10598-HNAY1).

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Co-Immunoprecipitation Assay, Incubation, Purification, Pull Down Assay, Construct

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet: In vitro differential suppression of podocyte IGF1R activity reveals that partial inhibition is beneficial but near total loss is highly detrimental (A) Representative Western blot shows >90% reduction of IGF1R protein in NC-IGF1RKD cells but no reduction of IR protein expression. Bar graphs show densitometry expressed as the mean fold change +/− SEM, t-test, ∗∗∗∗ p < 0.0001, n = 18 independent experiments. (B) Phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min was significantly reduced in NC-IGF1RKD podocytes. Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, ∗∗ p < 0.005, n = 3 independent experiments. (C) Western blot shows that IGF1R expression is reduced by ∼70% in wild-type podocytes exposed to 100 nM picropodophyllin for 24 h. Data are expressed as the mean ± SEM, t-test, ∗ p < 0.05, n = 3 independent experiments. No significant change in IR expression was observed. (D) Western blot shows the phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min in podocytes exposed to 100 nM picropodophyllin for 24 h. Data expressed as the mean ± SEM, ∗ p < 0.05, n = 3 independent experiments. (E) ∼50% of NC-IGF1RKD cells survive 7 days after gene excision. Treatment of wild-type podocytes with 100 nM picropodophyllin for 24 h has no effect on cell survival. Data are expressed as the mean ± SEM, t-test, ∗∗ p < 0.005, n = 3–4 independent experiments. See also Figures S5 and .

Article Snippet: Recombinant Mouse IGF1 protein , Novus , Cat# NBP2-35081.

Techniques: In Vitro, Activity Assay, Inhibition, Western Blot, Expressing, Comparison

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet:

Article Snippet: Recombinant Mouse IGF1 protein , Novus , Cat# NBP2-35081.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, RNAscope, Western Blot, Cell Culture, Software, Plasmid Preparation