Anti irs 1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-IRS-1 is a laboratory reagent used in research applications. It is a polyclonal antibody that specifically targets the IRS-1 (insulin receptor substrate-1) protein. IRS-1 is an important signaling molecule involved in insulin and growth factor signaling pathways. The Anti-IRS-1 antibody can be used to detect and study the expression and localization of IRS-1 in various cell and tissue samples.

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Spelling variants of this product

Anti-phospho-IRS-1 (Tyr-632; (3 citations) Mouse monoclonal anti‐human IRS‐1 (2 citations) Anti- p-IRS-1/2Y612 (2 citations)

10 protocols using anti irs 1

Western Blot Analysis of Metabolic Regulators

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Mouse liver, skeletal muscle or white adipose tissue were dissected and immediately frozen in liquid nitrogen. Whole-cell extracts were prepared using lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.2mM Na₃VO₄ and a protease inhibitor cocktail (Roche Diagnostics)). Protein concentration was measured by colorimetric assay (Bio-Rad Laboratories, Richmond, CA, USA) and equal amount of proteins was loaded onto SDS gels. After transfer to polyvinylidene difluoride membranes, proteins were probed with primary antibodies (1 μg/ml), followed by horseradish peroxidase-conjugated secondary antibodies, washed and visualized with SuperSignal West Pico/Dura chemiluminescent substrate (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). Blots were reprobed with β-actin-specific antibody for loading controls.
Anti-IRS-1 (Santa Cruz Biotechnology), anti-IRS-2 (Santa Cruz Biotechnology), anti-SCD-1 (Santa Cruz Biotechnology), anti-IRβ (Santa Cruz Biotechnology), anti-pAkt2 (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 (Santa Cruz Biotechnology), anti-ACCα (Santa Cruz Biotechnology), anti-FAS (Santa Cruz Biotechnology), anti-SREBP-2 (Santa Cruz Biotechnology), anti-HMGCR (Santa Cruz Biotechnology), anti-Albumin (Santa Cruz Biotechnology) and anti-β-actin (Santa Cruz Biotechnology) antibodies were used as primary antibodies.

Huo J., Ma Y., Liu J.J., Ho Y.S., Liu S., Soh L.Y., Chen S., Xu S., Han W., Hong A., Lim S.C, & Lam K.P. (2016). Loss of Fas apoptosis inhibitory molecule leads to spontaneous obesity and hepatosteatosis. Cell Death & Disease, 7(2), e2091-.

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Biochemical Markers of Cellular Stress

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Chemicals of analytical grade were purchased from Sigma (St Louis, MO, USA) except where stated otherwise. The following antibodies were used: anti-Atg7, anti-p62, anti-LC3 (light chain 3), anti-iNOS (inducible NO synthase), anti-CHOP (C/EBP homologous protein), anti-GRP78 (guaninenucleotide-releasing protein 78), anti-p-PERK (PKR-like ER kinase; Thr980) and anti-PERK (all from Cell Signaling Technology, Danvers, MA, USA); and anti-IRS-1 (insulin receptor substrate 1), anti-pY20, anti-GAPDH, peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated goat anti-mouse IgG (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Guo Q., Xu L., Li H., Sun H., Liu J., Wu S, & Zhou B. (2017). Progranulin causes adipose insulin resistance via increased autophagy resulting from activated oxidative stress and endoplasmic reticulum stress. Lipids in Health and Disease, 16, 25.

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Comprehensive Immunoblotting Antibody Panel

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Anti-PKR (sc-708, Santa Cruz), anti-peIF2alpha (44728G, Invitrogen), anti-eIF2alpha (#9772, Cell Signaling), anti-pJNK (#4668, Cell Signaling), anti-JNK (sc-7345, Santa Cruz), anti-pIRS1 (07–247, Millipore), anti-IRS1 (sc-559, Santa Cruz), anti-pIKKbeta (#2694, Cell Signaling), anti-IKKbeta (#2370, Cell Signaling), and anti-Tubulin (T9026, Sigma).

Taga M., Mouton-Liger F., Sadoune M., Gourmaud S., Norman J., Tible M., Thomasseau S., Paquet C., Nicoll J.A., Boche D, & Hugon J. (2018). PKR modulates abnormal brain signaling in experimental obesity. PLoS ONE, 13(5), e0196983.

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Insulin Signaling Pathway Analysis

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Anti-hnRNP A1, anti-ATF4, anti-CHOP, anti-Akt, anti-IRS-1, anti-Tubulin, and anti-GLUT4 were purchased from Santa Cruz. Anti-phospho-eIF2 (Ser51), anti-fatty acid synthase, anti-IR, anti-phospho-IR (Y1105/1151) anti-phospho-IRS-1 (Y986), anti-phospho-Akt (Ser473), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti-p44/42 MAPK (Erk1/2), anti-phospho-AMPKα (Thr172), anti-AMPKα, anti-phospho-Akt (Ser473), anti-phospho-IR (Y1105/1151), anti-IR, anti-GSK-3β (3D10), anti-GS, and anti-p-GS (Ser641) were purchased from Cell Signaling Technology. Anti-phospho-GSK-3β (Ser9) was purchased from Epitomics. Anti-Lamin B1 was purchased from Proteintech Group. Anti-Actin and anti-GAPDH were purchased from Abmart. Insulin was purchased from Wepon. 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-deoxyglucose) was purchased from Thermo Scientific. The siRNA-hnRNP A1 (m) and siRNA-Ctrl were purchased from Santa Cruz. TG test kit, Glu test kit, TC test kit, ALT, and AST test kits were purchased from Nanjing Jiancheng Bioengineering Institute. Insulin ELISA kit was purchased from ALPCO.

Zhao M., Shen L., Ouyang Z., Li M., Deng G., Yang C., Zheng W., Kong L., Wu X., Wu X., Guo W., Yin Y., Xu Q, & Sun Y. (2019). Loss of hnRNP A1 in murine skeletal muscle exacerbates high-fat diet-induced onset of insulin resistance and hepatic steatosis. Journal of Molecular Cell Biology, 12(4), 277-290.

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C2C12 Cell Lysis and Western Blot

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C2C12 cells were lysed in 20 mM Tris-HCl (pH 7.4), 5 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, and 1% NP-40 buffer supplemented with a protease inhibitor (1 µg/µL aprotinin, 1 µg/µL leupeptin, and 1 mM PMSF). Cell lysates were sonicated on ice twice for 15 seconds each time, and debris was removed by centrifugation (15,000 g) for 30 minutes at 4℃. Proteins (10 to 20 µg) were separated on a sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked with 5% skim milk in Tween-20-Tris-buffered saline (TBS-T) for 1 hour at room temperature, followed by incubation with primary antibody overnight at 4℃. The primary antibodies used were a total OXPHOS antibody cocktail (MitoScience, San Francisco, CA, USA), anti-IRS-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-pIRS-1 (Cell Signaling, Danvers, MA, USA), anti-Akt (Cell Signaling), anti-pAkt (Cell Signaling), anti-adenosine monophosphate (AMP)-activated protein kinase (AMPK; Cell Signaling), anti-phosphorylated AMPK (Cell Signaling), and anti-γ-tubulin (Sigma-Aldrich Co.). The secondary antibodies were anti-rabbit or anti-mouse immunoglobulin G.

Kim M.J., Koo Y.D., Kim M., Lim S., Park Y.J., Chung S.S., Jang H.C, & Park K.S. (2016). Rg3 Improves Mitochondrial Function and the Expression of Key Genes Involved in Mitochondrial Biogenesis in C2C12 Myotubes. Diabetes & Metabolism Journal, 40(5), 406-413.

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Insulin Signaling Pathway Analysis

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The muscle tissues were lysed on ice in a RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitor and 1 mM PMSF. After homogenization, the preparation was centrifuged at 20,000 g for 20 minutes at 4°C. The supernatant was then stored at -80°C for later analysis. The protein content in each sample was measured by the Bradford method. Equal amounts of lysate proteins (40 μg) were separated in 10% SDS PAGE gels before being transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk and then immunoblotted with antibodies 1 : 1000 anti-InsR, 1 : 1000 anti-IRS1 (phospho-Y612), 1 : 1000 anti-GLUT4, 1 : 500 pIRS1 (Ser 636), and 1 : 500 pIRS1 (Ser 639) (Santa Cruz) overnight at 4°C. After washes with 0.1% Tween 20 in TBS, the membranes were incubated with a horseradish peroxidase-linked secondary antibody. The protein bands were visualized after developing with a BCIP/NBT reagent. Protein content was normalized by monoclonal anti-β-actin (1 : 2000, Sigma-Aldrich).

Joseph A., Parvathy S, & Varma K.K. (2021). Hyperinsulinemia Induced Altered Insulin Signaling Pathway in Muscle of High Fat- and Carbohydrate-Fed Rats: Effect of Exercise. Journal of Diabetes Research, 2021, 5123241.

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Western Blot Analysis of Signaling Proteins

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Differentiated 3T3-L1 cells were harvested and proteins were extracted with lysis buffer (PRO-PREP; Intron Biotechnology, Seoul, Republic of Korea) for 60 min at 4°C. Protein samples (30 μg) were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences, Westborough, MA, USA) and probed with the indicated primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, USA). The signals were detected using enhanced chemiluminescence (ECL) kits (Amersham Biosciences). Anti-phospho Akt (Ser473; 1:1,000), anti-Akt (1:1,000), anti-phospho AMPK (Thr172; 1:1,000), anti-AMPK (1:2,500), anti-phospho NFκBp65 (Ser536; 1:1,000), anti-NFκBp65 (1:2,500), anti-phospho IκB (Ser32; 1:1,000) and anti-IκB (1:1,000) antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-phospho IRS-1 (Tyr632; 1:1,000), anti-IRS-1 (1:1,000), anti-HO-1 (1:1,000) and anti-β-actin (1:5,000) were obtained from Santa Cruz Biotechnology.

Kwon C.H., Sun J.L., Kim M.J., Abd El-Aty A.M., Jeong J.H, & Jung T.W. (2020). Clinically confirmed DEL-1 as a myokine attenuates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes via AMPK/HO-1- pathway. Adipocyte, 9(1), 576-586.

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Investigating Insulin Receptor Signaling Pathways

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Human embryonic kidney 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The full-length human IR cDNA (exon 11- form) was cloned into plasmid pCMV-HA (Clontech). Site-directed mutations were introduced using QuikChange.
Cells were transfected with the appropriate plasmids using X-treme GENE HP DNA transfection reagent (Roche), according to the manufacturer’s instructions. Whole-cell lysates were prepared using a buffer containing 1% NP40, 25 mM Tris-HCl, pH 7.5, 100 mM NaCl and 1 mM EDTA. The following antibodies were used for western blotting: anti-phosphotyrosine, clone 4G10 (cat. no. 05–321, Millipore) at 1:2,000 dilution, anti-IRβ, C-19 (cat. no. sc-711, Santa Cruz Biotechnology) at 1:1,000 dilution, anti-IRS-1 (cat. no. 07–843, Millipore) at a concentration of 0.75 μg ml ⁻¹ and anti-phospho-IR (pTyr1162/pTyr1163; cat. no. 44804G, Life Technologies) at 1:5,000 dilution. Subsequently, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. After washing several times, the membranes were developed with an enhanced chemiluminescence (ECL) system according to the manufacturer’s instructions (Thermo Scientific).

Cabail M.Z., Li S., Lemmon E., Bowen M.E., Hubbard S.R, & Miller W.T. (2015). The insulin and IGF1 receptor kinase domains are functional dimers in the activated state. Nature communications, 6, 6406.

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Insulin Signaling Pathway Regulation

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Dulbecco's modified Eagle's medium (DMEM) was purchased from Flow Laboratories (Irvine, UK); glutamine and antibiotics were from Hyclone (Cramlington, UK). Insulin was from Sigma (St Louis, MO, USA). PCA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Electrophoresis reagents were from Bio-Rad (Hercules, CA, USA). Anti-IRS-1 (catalog number sc-7200), anti-GLUT4 (catalog number sc-53566), anti-p65 NF-κB (catalog number sc-8008), IL-1β (catalog number sc-52865) and horseradish peroxidase-conjugate anti-goat (catalog number sc-7020), and anti-rabbit (catalog number sc-2357) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugate anti-mouse (catalog number 172-1011) antibody was from Bio-Rad (Kidlington, UK). Anti-Akt (catalog number 2920S), anti-phospho Akt Ser 473 (catalog number 4060S), anti-IRβ (catalog number 81764), anti-phospho p65 NF-κB Ser536 (catalog number 3033S) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Antibody against PTP1B (catalog number Ab2009) was purchased from Abcam (Cambridge, United Kingdom). Anti-phospho Tyr 608 IRS-1 (catalog number 09-432) and anti-β-actin (catalog number MABT826) antibodies were from Merck-millipore (Temecula, CA, USA). ELISA Kit for IL-6 was from Biolegend (San Diego, CA, USA) and Tyrosine Phosphatase Assay System from Promega (Madison, WI, USA).

Ormazabal P., Scazzocchio B., Varì R., Santangelo C., D'Archivio M., Silecchia G., Iacovelli A., Giovannini C, & Masella R. (2018). Effect of protocatechuic acid on insulin responsiveness and inflammation in visceral adipose tissue from obese individuals: possible role for PTP1B. International journal of obesity (2005), 42(12).

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Autophagy, ER Stress, and Insulin Signaling

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The following antibodies were used: anti-Atg7, anti-p62, anti-LC3, anti-p-PERK, anti-PERK, anti-p-eIF2A and anti-eIF2A (Cell Signaling Technology Inc. Danvers, MA); anti-IRS-1, anti-pY20, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR, anti-PGRN, anti-GAPDH (Santa Cruz Biotechnology, Inc.).

Zhou B., Li H., Liu J., Xu L., Guo Q., Sun H, & Wu S. (2015). Progranulin induces adipose insulin resistance and autophagic imbalance via TNFR1 in mice. Journal of molecular endocrinology, 55(3).

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