3-isobutyl-1-methylxanthine (IBMX, I5879), dexamethasone (D8893), human insulin solution (I9278), antibody to hypoxia-inducible factor (HIF-1α, H6536) and sodium sulfite (Na2SO3, S0505) were obtained from Sigma-Aldrich. Antibodies against glucose transporter 1 (Glut1; ab652), glucose transporter 4 (Glut4; ab654), Akt (ab8805), S6K (ab9366), phosphorylated Akt Thr308 (pAkt, ab38449), phosphorylated pyruvate dehydrogenase E1 Ser293 (pPDHE1, ab92696), and β-actin (ab6276) were obtained from Abcam (Cambridge, UK). Antibodies to pyruvate dehydrogenase E1 (PDHE1, sc-65242), insulin receptor β (IRβ, sc-711), insulin receptor substrate-1 (IRS-1, sc-7200), GSK-3β (sc-7291), SREBP1 (sc-13551) and phosphorylated GSK3β (pGSK3β; sc-11757-R) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to phosphorylated AktSer473 was purchased from Calbiochem (Gibbstown, NJ). Antibodies to AMPK (#2603), phosphorylated AMPKα (Thr172, pAMPK, #2535), and phosphorylated acetyl CoA carboxylase (pACC, #3661) were purchased from Cell Signaling (Beverly, MA). Antibody to phosphorylated IRS-1 Ser-307 (28863) was purchased from Upstate Biotechnology (Canastota, NY). PPARγ (MAB3872) antibody was from EMD Millipore (Billerica, MA, USA).
Sc 711
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-711 is a laboratory equipment product manufactured by Santa Cruz Biotechnology. It is designed for use in scientific research and analysis. The core function of the Sc-711 is to [BRIEF DESCRIPTION OF CORE FUNCTION].
Automatically generated - may contain errors
Lab products found in correlation
Ab9366, by Abcam (2 mentions)
Ab38449, by Abcam (2 mentions)
Ab654, by Abcam (2 mentions)
Ab652, by Abcam (2 mentions)
Ab8805, by Abcam (2 mentions)
Sc 65242, by Santa Cruz Biotechnology (2 mentions)
Sc 7200, by Santa Cruz Biotechnology (2 mentions)
Pgsk3β sc 11757 r, by Santa Cruz Biotechnology (2 mentions)
Pparγ mab3872 antibody, by Merck Group (2 mentions)
Sc 7291, by Santa Cruz Biotechnology (2 mentions)
Sc 13551, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Sc 2030, by Santa Cruz Biotechnology (1 mentions)
Ab51074, by Abcam (1 mentions)
Sc 358914, by Santa Cruz Biotechnology (1 mentions)
Anti pakt 4060, by Cell Signaling Technology (1 mentions)
Pxi 4 imager, by Syngene (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ab58968, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
14 protocols using sc 711
1
Insulin Signaling Pathway Regulation
2
Insulin Receptor Signaling Pathway Analysis
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The antibodies used in this study include the following: anti-MT1-MMP antibody (ab51074, Abcam; 1:2000 for western blotting); anti-insulin Rα antibody (sc-57344, Santa Cruz, 1:2000 for western blotting); anti-insulin Rβ antibody (sc-711, Santa Cruz, 1:2000 for western blotting); anti-insulin Rβ antibody (clone CT-3, MAB S65, millipore, 1:2000 for western blotting); anti-Akt (4685, Cell Signaling, 1:3000 for western blotting): anti-pAkt (4060, Cell Signaling, 1:2000 for western blotting); anti-β-actin (12262, Cell Signaling, 1:5000 for western blotting); goat anti-rabbit antibody conjugated with HRP (sc-2030, Santa Cruz, 1:2000); Rabbit anti-mouse antibody conjugated with HRP (sc-358914, Santa Cruz, 1:2000).
3
Insulin Signaling Pathway Analysis in Tissues
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Studies were performed as described (30 (link)–32 (link)). After ND or HFD feedings and ASO treatments as shown in Fig. 1A , mice were fasted overnight, then injected ip with 100 μl saline or 5 U/kg Humulin-R. Exactly 10 min after injection, mice were euthanized, and liver, eWAT, and gastrocnemius muscle biopsies were flash-frozen in liquid nitrogen before protein extraction. Fifty micrograms of protein were resolved in precasted 4–12% Bis–Tris gels (Life Technologies, Carlsbad, CA), transferred to polyvinylidene difluoride membranes, and probed with antibodies for protein kinase B (AKT) (Cell Signaling 9272; 1:1,000 dilution; Danvers, MA), phospho-AKT (Cell Signaling 9271; 1:1,000 dilution), insulin receptor (IR) (Santa Cruz sc-711; 1:1,000 dilution; Dallas, TX), phospho-IR (Cell Signaling 3024; 1:1,000 dilution), and β-actin (Thermo RB-9421; 1:1,000 dilution; Carlsbad, CA), as described (30 (link)–32 (link)), in TBS-Tween20 containing 5% nonfat dry milk. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies [Bio-Rad 170-6515 (anti-rabbit), 170-6516 (anti-mouse); 1:5,000 dilution; Hercules, CA], using a high-resolution PXi-4 imager (SynGene, Frederick, MD). Band intensities were calculated using GeneTools version 4.03 software (SynGene).
4
Western Blot Analysis of Autophagy and Insulin Signaling
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The procedure of western blot analysis was carried out as described previously (Delfin et al., 2011 (link)). The following primary antibodies were used: antibodies for SQSTM1/p62 (5114; Cell Signaling), LC3B (ab48394; Abcam), Beclin-1 (SC-11427; Santa Cruz), IR (sc-711; Santa Cruz) and nephrin (ab58968; Abcam) Primary antibody against β-actin and horseradish peroxidase-conjugated secondary antibodies were from ZSGB-BIO.
5
Insulin and AMPK Signaling Pathway Analysis
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For the analyses of insulin and AMPK signal transduction, mice that had been denied access to food overnight received DS20060511 (10 mg kg−1), insulin (5 U kg−1), or saline (vehicle control) via the inferior vena cava under isoflurane anesthesia and 10 min later, the triceps surae muscle specimen was excised for western blot analysis. The tissue sample was homogenized with a Polytron homogenizer and lysed in a lysis buffer (25 mM Tris-HCl, pH 7.4, 100 mM NaF, 50 mM Na4P2O7, 10 mM EGTA, 10 mM EDTA, 10 mM Na3VO4, 1 mM PMSF, and 1% NP-40) on ice, and the lysate was centrifuged at 17,860 × g for 10 min at 4 °C. The supernatant was collected and the protein concentrations were determined by BCA assay (Thermo Fisher Scientific). Immunoprecipitation of IRβ and IRS1 was performed as described previously10 (link), using specific antibodies against IRβ (sc-711, Santa Cruz Biotechnology, Dallas, TX), IRS1 (06-248, Merck Millipore, Burlington, MA), and for detection of phosphotyrosine (05-321, Merck Millipore). Five milligrams of the extracts were incubated with specific antibodies against IRS1 for 1 h at 4 °C. Then, protein G-Sepharose was added, followed by incubation for 2 h at 4 °C. After washing three times, the immunocomplexes were subjected to immunoblotting.
6
Insulin Signaling Pathway Regulation
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3-isobutyl-1-methylxanthine (IBMX, I5879), dexamethasone (D8893), human insulin solution (I9278), antibody to hypoxia-inducible factor (HIF-1α, H6536) and sodium sulfite (Na2SO3, S0505) were obtained from Sigma-Aldrich. Antibodies against glucose transporter 1 (Glut1; ab652), glucose transporter 4 (Glut4; ab654), Akt (ab8805), S6K (ab9366), phosphorylated Akt Thr308 (pAkt, ab38449), phosphorylated pyruvate dehydrogenase E1 Ser293 (pPDHE1, ab92696), and β-actin (ab6276) were obtained from Abcam (Cambridge, UK). Antibodies to pyruvate dehydrogenase E1 (PDHE1, sc-65242), insulin receptor β (IRβ, sc-711), insulin receptor substrate-1 (IRS-1, sc-7200), GSK-3β (sc-7291), SREBP1 (sc-13551) and phosphorylated GSK3β (pGSK3β; sc-11757-R) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to phosphorylated AktSer473 was purchased from Calbiochem (Gibbstown, NJ). Antibodies to AMPK (#2603), phosphorylated AMPKα (Thr172, pAMPK, #2535), and phosphorylated acetyl CoA carboxylase (pACC, #3661) were purchased from Cell Signaling (Beverly, MA). Antibody to phosphorylated IRS-1 Ser-307 (28863) was purchased from Upstate Biotechnology (Canastota, NY). PPARγ (MAB3872) antibody was from EMD Millipore (Billerica, MA, USA).
7
Western Blot Analysis of Insulin Signaling Proteins
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Tissues were homogenized as described (Brüning et al., 1998 (link)). Western blot analyses of insulin signalling proteins were performed on liver homogenates as previously described (Valverde et al., 1998 (link)). For immunoprecipitation, after protein content determination, equal amounts of protein (400 μg) were immunoprecipitated overnight at 4°C with anti-GLUT-2 antibodies (C-19) (1:500; sc-7580, Santa Cruz Biotechnology, Dallas, TX). The immune complexes were collected on protein A-agarose beads and submitted to SDS-PAGE. For western blot, the antibodies used were anti-insulin receptor β subunit (1:1000; sc-711), anti-GLUT-2 (H-67) (1:500; sc-9117) and anti-IGF-I (1:200; sc-9013) (all from Santa Cruz Biotechnology) and anti-β-actin (1:5000; A2228, Sigma-Aldrich, St. Louis, MO). Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated polyclonal anti-rabbit or mouse antibodies, respectively (1:5000; NA931V and NA934V, GE Healthcare, Buckinghamshire, UK). Loading was normalized by β-actin. The band intensities were quantified using ImageJ software (http://rsb.info.nih.gov/ij ).
8
Western Blot Analysis of IGF1R and INSR
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Proteins separated on SDS-PAGE gels were transferred to polyvinylidene difluoride membranes (Biorad). Membranes were blocked in PBS-Tween with 5% nonfat dry milk. Primary antibodies for detection of IGF1R (Cell Signaling 9750) and INSR (Santa Cruz SC711) were used at 1:1000 dilution. Bound primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (1:3000, Santa Cruz Biotechnology) and chemiluminescent substrate (ECL plus, Thermo Scientific).
9
Immunoprecipitation and Immunoblotting of Insulin Signaling Proteins
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To prepare the lysates, the cells and tissues were homogenized in buffer A (25 mM Tris-HCl, pH 7.4, 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM EGTA and 1 mM phenylmethylsulfonyl fluoride). For immunoprecipitation of Irβ, Irs1 and Irs2, the lysates were incubated with rabbit polyclonal antibody against Irβ (sc-711, Santa Cruz), Irs1 (06–248, Millipore) or Irs2 (3089, Cell Signaling) for 1 h at 4 °C. Then, protein G-Sepharose was added, followed by incubation for a further 1 h at 4 °C. Thereafter, after washing 3 times with buffer A, the immunocomplexes were resolved on 7% or 10% SDS-PAGE. Phosphorylated or total protein was analyzed by immunoblotting using specific antibodies against Irβ, Irs1, Irs2 and phosphotyrosine. Phosphorylated or total protein of Akt, FoxO1 and STAT6 was isolated by immunoblotting using specific antibodies after the tissue lysates were resolved by SDS-PAGE and transferred to a Hybond-P PVDF transfer membrane (Amersham Biosciences). Bound antibodies were detected with HRP-conjugated secondary antibodies using ECL detection reagents (Amersham Biosciences). Uncropped scans of the blots can be found as a Supplementary Figure in the Supplementary Information (Supplementary Fig. 10 and Supplementary Fig. 11 ).
10
Immunohistochemical Detection of Insulin Receptor
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Immunohistochemistry was performed as described previously [21] (link). Briefly, an avidin-biotin peroxidase system (Vector Lab, Burlingame, CA, USA) was used to detect anti-insulin receptor rabbit polyclonal antibody (#SC-711: Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Reactions were then visualized with diaminobenzidine (DAB) as substrate (Vector Lab, Burlingame, CA, USA). The sections were counterstained with hematoxylin. All staining procedures were performed as described by the manufacturer.
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