The primary antibodies used were anti-β-MHC (1:1000, Sigma-Aldrich), anti-phospho-Akt (Ser473) (1:1000, Cell Signaling), anti-Akt (1:1000, Cell Signaling), anti IRS-1 (1:1000, Cell Signaling), anti-phospho-IRS-1 (Ser307) (1:500, Cell Signaling) and anti-MCU (1:2000, Abcam). Then the blots were incubated with a horseradish peroxidase-coupled secondary antibody (1:5000, Pierce). ImageJ software was used for image densitometry.
Anti phospho irs 1 ser307
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-IRS-1 (Ser307) is a laboratory reagent used for the detection and quantification of phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307. It is a specific antibody that binds to the phosphorylated form of IRS-1 at this particular serine residue.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (3 mentions)
Anti irs1, by Cell Signaling Technology (3 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Anti mcu, by Abcam (1 mentions)
Anti β mhc, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phosstop, by Roche (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Recombinant human insulin, by Eli Lilly (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti g6pase, by Santa Cruz Biotechnology (1 mentions)
Anti jnk, by Santa Cruz Biotechnology (1 mentions)
Atorvastatin, by Pfizer (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
2 n 7 nitrobenz 2 oxa 1 3 diazol 4 yl amino 2 deoxy d glucose 2 nbdg, by Thermo Fisher Scientific (1 mentions)
Recombinant human insulin, by Merck Group (1 mentions)
5 protocols using anti phospho irs 1 ser307
1
Immunoblotting Analysis of Key Signaling Proteins
2
Quantitative Western Blot Analysis of Insulin Signaling
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As described previously [4 (link)], gastrocnemius muscle and the liver samples were processed by homogenizing in ice-cold RIPA buffer (Beyotime P0013C, China) supplemented with complete protease inhibitor cocktail (Roche, Germany) and PhosSTOP (Roche, Germany). The protein concentration of the supernatant obtained by centrifugation was determined with a BCA protein assay kit (Pierce Biotechnology, Rockford). The protein extracts (40 μg) for each preparation were separated by SDS-PAGE and analyzed by specific antibodies including anti-AKT (Cell Signaling Technology, Cat no. #4685, Beverly, MA, USA), anti-phospho-AKT Ser473 (Cell Signaling Technology, Cat no. #4058, Beverly, MA, USA), anti-IRS1 (Cell Signaling Technology, Cat no. #2382, Beverly, MA, USA), and anti-phospho-IRS1 (Ser307) (Cell Signaling Technology, Cat no. #2381, Beverly, MA, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies, the immunoblots were visualized with an ECL Kit (WBKLS0050; Millipore, Billerica, MA, USA), and quantitated by densitometric analysis using ImageJ.
3
Antibody Immunoblotting Protocol
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The antibodies used were anti-Akt (rabbit polyclonal, #9272), anti-phospho-Akt (rabbit polyclonal, #9271), anti-β-Actin (rabbit polyclonal, #4967), anti-IRS1 (rabbit polyclonal, #2382) and anti-phospho-IRS1ser307 (rabbit polyclonal, #2381) from Cell Signaling Technology (Beverly, MA, USA); anti-JNK (rabbit polyclonal, SC1648), anti-phopho-JNK (mouse monoclonal, SC6254), anti-G6pase (goat polyclonal, SC 27198) and anti-α-Tubulin (mouse monoclonal, SC8035) from Santa Cruz Technology (Santa Cruz, CA, USA). Atorvastatin was obtained from Pfizer (Loughbeg, County Cork, Ireland) and Diacerein was kindly provided by TRB-Pharma (Campinas, Brazil). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, Indiana, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless specified elsewhere.
4
Comparative Evaluation of Insulin Sensitizers
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RSV (purity > 99%) and human recombinant insulin were obtained from Sigma-Aldrich (St. Louis, MO, USA). R3G (purity > 98%) and R4G (purity > 95%) compounds were procured from Bertin Pharma (Montigny-le-Bretonneux, France). All three chemicals were dissolved in dimethyl sulfoxide and the solutions were stored in −20 °C. 2-[N-(7-Nitrobenz-2-oxa1,3-diazol-4-yl) amino]-2-deoxy-d -glucose (2-NBDG) was purchased from Life Technologies (Carlsbad, CA, USA). The antibody to β-actin was obtained from Abcam (Cambridge, UK), while anti-AMPKα, anti-AMPKα (Thr172), anti-IRS-1, anti-phospho-IRS-1 (Ser307) and anti-phospho-IRS-1 (Ser612) rabbit anti-human polyclonal antibodies were purchased from Cell Signaling Technology. The antibody to Phospho-IRS-1 (Tyr608) was obtained from Millipore (Billerica, MA, USA). All chemicals used in our study were of analytical grade.
5
Cardiac Tissue Protein Analysis
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Samples of cardiac tissue protein were prepared using a protein Extraction Kit (Solarbio, Beijing, China). Protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay kit (Solarbio, Beijing, China). The following primary antibodies were used in this study: anti-Rheb (Abcam, ab25873), anti-S6K2 (Cell Signaling, 14130), anti-Rictor (Cell Signaling, 2140), anti-phospho-IR (Abcam, ab62321), anti-phospho-IRS-1 (Ser 307) (Cell Signaling, 2381), anti-mTOR (phospho S2448) (Abcam, ab109268/R), anti-mTOR (Abcam, ab32028), and anti-GAPDH (ZSGB-BIO, TA-08/M). Protein samples were separated on 6% (for Rictor and p-IRS-1) or 12% (for S6K2, Rheb, and GAPDH) SDS-PAGE gels, and were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk, after which they were incubated with a 1:1000–1:2000 dilution of primary antibodies overnight at 4 °C. Subsequently, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) for 1 h at room temperature (about 25 °C). The enhanced chemiluminescence (ECL) method was utilized to visualize the protein bands.
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