44-816G

antibody from Invitrogen Antibodies

Targeting: IRS1 HIRS-1

Provider product page for 44-816G

Western blot

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [46]
Comments [0]
Validations
Western blot [1]
Flow cytometry [1]
Other assay [23]

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Product number: 44-816G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-IRS1 (Tyr612) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: On western blotting this antibody detects a protein with the apparent MW of 165 kDa. Positive controls used were Insulin (50 nM for 5 minutes) or TPA (100 ng/mL for 30 min) treated human embryonic kidney cells (293T) or Chinese Hamster Ovary cells (CHO-T) transiently transfected with a plasmid encoding human IRS1.
Reactivity: Human
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

IRS1 protein structure - 44-816G shown in red.

Supportive validation

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Supportive validation

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Experimental details: Fig. 4 Localisation of signalling components in human astrocytes. a Immunocytochemistry demonstrates nuclear localisation of IRS1 phosphorylated at serine 616 (S616) and S636/639 but not total IRS1 b Representative immunoblots of fractionated cell lysates showing the localisation of total IRS1 and IRS1 phosphorylated at S616 and tyrosine 612 (Y612) by immunoblot. Loading control for cytosplasmic fraction are EEF2 and for nuclear fraction is SP1. Molecular weight markers are indicated (kDa)

Supportive validation

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Experimental details: Figure 2 FTI-277 treatment reversed burn-induced impaired insulin receptor (IR)-insulin receptor substrate-1 (IRS-1) signaling in skeletal muscle. IR protein expression was not altered by burn, FTI-277 or insulin (B). Insulin-stimulated IR phosphorylation was attenuated at 3 days after burn compared with sham-burned mice, which was almost completely restored by FTI-277 treatment (C, D). Burn decreased IRS-1 protein expression (E) and insulin-stimulated IRS-1 phosphorylation (F, G), both of which were reversed by FTI-277. Insulin significantly increased p-IRS-1/GAPDH ratio and p-IRS-1/IRS-1 ratio in sham-burned mice and FTI-277-treated burned mice, whereas insulin failed to significantly increase p-IRS-1/GAPDH ratio or p-IRS-1/IRS-1 ratio in vehicle-treated burned mice (F, G). There was a trend of increase in insulin-stimulated p-IRS-1/IRS-1 ratio in vehicle-treated burned mice, but there was no statistical difference (G). n = 5 mice per group for saline-injected mice, n = 6 mice per group for insulin-injected sham-burned mice, n = 8 mice per group for insulin-injected burned mice. *p

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Experimental details: Figure 1 NT219 inhibits the IGF1R to AKT signaling pathway in human melanoma cells. (A) Chemical formula of NT219. (B-C) NT219 treatment reduces the IGF-induced autophosphorylation of the IGF1R, induces inhibitory Ser-phosphorylation and subsequent elimination of the insulin receptor substrate 1 (IRS1) and 2 (IRS2), and prevents the IGF1-induced activation of AKT in A375 human melanoma cells, following short (B) and long (C) exposures. (D) Temporal analysis of the effects of NT219 on components of the IGF pathway indicates that the compound acts on the IGF1R and AKT within 1 h to block the signaling cascade and confers Ser-phosphorylation and degradation of IRS1 and IRS2 2-8 h after exposure to achieve long-term inhibition. (E) NT219 elevates the expression levels of the FOXO target genes p27 and manganese superoxide dismutase (MnSOD) in A375 cells.

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Experimental details: Fig 3 Effect of insulin-like growth factor 1 receptor (IGF1R) siRNAs on activity of ZSTK474 to inhibit cell proliferation and phosphatidylinositol 3-kinase downstream signaling. (a) Knockdown of IGF1R protein expression using IGF1R siRNAs. MKN28, St-4, SNB75, and PC3 cells were transfected with control siRNA (si-cont.) or IGF1R siRNAs (siIGF1R-1 or siIGF1R-2) and the expression of IGF1R protein was measured by immunoblot analysis. (b) Dose-response curves of ZSTK474 against cell growth in MKN28, St-4, SNB75, and PC3 cells after transfection with control siRNA or IGF1R siRNAs. Cell growth was assessed by sulforhodamine B assay. Assays were carried out in duplicate and the data are representative of two independent experiments. (c) MKN28, St-4, SNB75, and PC3 cells were transfected with either control siRNA or IGF1R siRNA, and then exposed to ZSTK474 at the indicated concentrations for 3 h. Cells were then harvested and immunoblot analyses of indicated proteins were carried out.

Supportive validation

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Experimental details: Figure 5 Effect of DCI on insulin receptor (IR) phosphorylation and the IRS-1/AKT pathway in alpha-TC1-6 cells exposed to palmitate. Immunoprecipitation and Western blot analysis for total IR (Tyr1150/1151-beta subunit) (p-IR-beta) and total IR; Western blot analysis for p-IRS-1 (Tyr612), total IRS-1 (IRS-1), p-AKT (Ser 473), total AKT (AKT), and beta-actin: alphaTC1-6 cells were treated with DCI (0.1 or 1 mM for 48 h) in the presence or absence of palmitate (0.5 mM for 48 h) and acutely stimulated with insulin (10 -9 M for 5 min). ( A ) Representative immunoblot of three independent experiments. ( B ) Means +- SEM of densitometric analysis. One-way ANOVA followed by Bonferroni test ( n = 3) was used to evaluate statistical significance in insulin-stimulated cells treated with increasing concentrations of DCI with respect to insulin-stimulated controls in the absence and presence of palmitate: * p < 0.05, ** p < 0.01, *** p < 0.001. n.s., not significant; DCI, D-chiro-inositol.

Supportive validation

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Experimental details: Figure 2 Exendin-4 improves insulin signaling in a neuron under palmitic acid-induced oxidative stress. ( A ) Comparison of mRNA expression levels of insulin signaling related genes ( FOXO1 , SLC2A4 , and RPS6KB1 ) in SH-SY5Y cells treated with vehicle (C or Ctr), Exendin-4 (E or Ex-4), palmitic acid (P or PA), and both palmitic acid and Exendin-4 (PE or PA+Ex-4). The mRNA level of each gene is normalized to GAPDH level. ( B ) Western blot of insulin signaling through 20 nM insulin stimulation for 10 min in SH-SY5Y cells treated with reagents described in A. Each protein level is normalized to beta-actin. The pIRS-1, pAKT, and pGSK-3beta levels were normalized to IRS-1, AKT, and GSK-3beta of total form. ( C , D ) immunocytochemistry images of pIRS-1 expression in SH-SY5Y cells and PCN neurons at DIV 7 treated with reagents described in A. Nuclei were counterstained with DAPI (blue) and insulin receptor stained with pIRS-1 (green), >100 ( E ) and >50 ( F ) cells per group were analyzed. Scale bar: 50 mum. Data information: SH-SY5Y cells were treated with 10 nM of Exendin-4 and 50 muM of palmitic acid per group according to the treatment plan ( Figure S1B,C ). In ( A - D ), error bars represent S.E.M. from three independent experiments. n.s p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two-tail t -tests with Welch's correction).

Supportive validation

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Experimental details: FIGURE 4 Sunitinib inhibits insulin signaling pathway in mesenteric arteries in rats. (A) Representative western blots of total IRS-1 and phospho-Tyr612 IRS-1 in mesenteric artery tissues isolated on day 7 from control and sunitinib-treated rats, without insulin stimulation; bar graph represents phospho-Tyr 612 IRS-1 levels normalized to total IRS-1 levels (lower panel) ( p = 0.007 vs. control; n = 6 rats/group). (B) Representative western blots of total AKT and phospho-Ser473 AKT in mesenteric artery tissues isolated on day 7 from control and sunitinib-treated rats, without insulin stimulation; bar graph represents phospho-Ser473 AKT levels normalized to total AKT levels (lower panel) ( p = 0.002 vs. control; n = 7 rats/group). (C) Representative western blots of total eNOS and phospho-Ser1177 eNOS in mesenteric artery tissues isolated on day 7 from control and sunitinib-treated rats, without insulin stimulation; bar graph represents phospho-Ser1177 eNOS levels normalized to total eNOS levels (lower panel) ( p = 0.005 vs. control, n = 7 rats/group). (D) Representative western blot images of total IRS-1 and phospho-Tyr 612 IRS-1 in mesenteric artery tissues dissected on day 7 from insulin-injection control rats or insulin-injection rats treated with sunitinib; bar graph represents phospho-Tyr612 IRS-1levels normalized to total IRS-1 levels (lower panel) ( p = 0.016 vs. control; n = 7 per group). (E) Representative westerns of total AKT and phospho-Ser473 AKT in mesenteric art

Supportive validation

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Experimental details: FIGURE 5 Sunitinib inhibits insulin signaling associated molecules in rat MAECs (A) Representative western blots of total IRS-1 and phospho-Tyr612 IRS-1 in control MAECs or in MAECs treated with 1 muM sunitinib for 30 min and bar graph represents phospho-Tyr612 IRS-1 levels normalized to total IRS-1 levels. (B) ( p = 0.015 vs. control; n = 6 batches of cells in each group). (C) Representative western blot images of total AKT and phospho-Ser473 AKT expression in control MAECs cells or in MAECs treated with 1 muM sunitinib for 30 min and bar graph represents phospho-Ser473 AKT levels normalized to total AKT levels. (D) ( p = 0.003 vs. control; n = 7 batches of cells in each group). (E) Representative western blot images of total eNOS and phospho-Ser1177 eNOS expression in control MAECs cell or in MAECs treated with 1 muM sunitinib for 30 min and bar graph represents phospho-Ser1177 eNOS levels normalized to total eNOS levels. (F) ( p = 0.002 vs. control, n = 7 batches of cells in each group). (G) The concentrations of nitrate and nitrite measured in the control MAECs cells or in MAECs treated with 1 muM sunitinib for 30 min, reflecting NO production under indicated experimental condition ( p < 0.001 vs. control; n = 6 per group).

Supportive validation

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Experimental details: Figure 6 Restoration of IRS-1 signaling corrects Kir4.1 levels. (A) rMC-1 cells were treated with 10 nM insulin, Western blot showing an increase in phosphorylation of IRS-1 Tyr612 , Akt Ser473 , BMAL-1, and Kir4.1. (B) Line chart showing quantification of the respective protein. The retinal explants from db/m and db/db mice were treated with insulin. (C) Western blot. (D) Bar chart showing an increase in phosphorylation of IRS-1 Ser307 . (E) Western blot showing IRS-1 Tyr612 phosphorylation after insulin treatment and (F) quantification of phospho-IRS-1 Tyr612 . (G) Western blot showing a concurrent increase in BMAL1 and phosphorylation of Akt Ser473 . (H) Bar chart showing a quantification of phospho-Akt Ser473 and (I) BMAL1. (J) Western blot showing Kir4.1 protein levels in insulin-treated explants. (K) Bar chart showing the quantification of Kir4.1 (n = 5).