The proteins in the supernatant of the centrifuged samples were separated by SDS/PAGE and transferred on to nitrocellulose membranes after blocking in skim milk for 1 h. The blots were then incubated with the indicated anti-bodies: rabbit anti-rat adiponectin, rabbit anti-rat adiponectin receptor 1 (AdipoR1; 1:1000; Cell Signaling, Davers, MA), mouse anti-rat adiponectin receptor 2 (AdipoR2; 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), rabbit anti-rat phosphorylated AMPK, rabbit anti-rat AMPK, rabbit anti-rat phospho-AMPK (1:1000; Cell Signaling), and rabbit anti-rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500; Millipore, Darmstadt, Germany). The specificity of the antibody was determined by mixing the blocking peptide with the corresponding antibody at a ratio of 10:1 overnight in an oscillator in 4°C, and then incubated with IRDye secondary antibody (Li-COR, Lincoln, NE). The bands were detected by the Odyssey Infrared Imaging System (Li-COR). The densities of the bands were semi-quantified and analyzed by the Odyssey Application Software (Li-COR). The amount of protein transferred on ghnto the membranes was verified by immunoblotting for GAPDH.
Adipor1
Manufactured by Cell Signaling Technology
Sourced in United States
AdipoR1 is a laboratory equipment product from Cell Signaling Technology. It is a receptor that binds the adiponectin protein, which is involved in regulating glucose and lipid metabolism. The core function of AdipoR1 is to facilitate the detection and study of the adiponectin receptor in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Odyssey application software, by LI COR (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Gapdh, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
P ampk, by Santa Cruz Biotechnology (1 mentions)
Flotillin 1, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Sds page gel, by Merck Group (1 mentions)
Ecl detection system, by Promega (1 mentions)
Tbs t, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
4 protocols using adipor1
1
Adiponectin Signaling Pathway Analysis
2
Protein Extraction and Western Blot Analysis
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Cells were lysed, and then total proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer. Total proteins were analyzed with an electrophoresis method using sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, and then transferred to a polyvinylidene fluoride (PVDF) membrane with a 0.45-μm pore size (Roche, Branchburg, NJ, USA). This process was prohibited with 5% skimmed milk and the preparation then washed three times with Tris-buffered saline (TBST) at room temperature, and then at 4 °C, and probed with the antibodies: AdipoR1, SIRT-1, AMPK (1:4000, Cell Signaling, Danvers, MA, USA), p-AMPK, Flotillin-1, β-actin (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. Finally, the preparation was incubated at room temperature with proper secondary antibodies (1:5000 dilutions, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3
Western Blot Analysis of Adiponectin Signaling
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Cells were collected and total proteins were extracted in 40 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Triton X-100 (Sigma, USA), supplemented with protease inhibitors. Protein concentration was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Equal amounts of protein were resolved on 10% SDS-PAGE gels (Sigma, USA), and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk in TBST (Sigma, USA), then probed with antibodies against AdipoR1, SIRT-1, AMPK (1: 1000) (Cell Signaling Technology, Danvers, MA, USA), p-AMPK, TLR-4 (1: 1000) (Santa Cruz, CA, USA), β-actin (1: 2000) (Cell Signaling Technology, Danvers, MA, USA), respectively, at 4°C, overnight. After three times washes, blots were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Cell Signaling Technology, Inc., Boston, MA, USA). Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) detection system (Promega, USA). The protein levels of the stripes were normalized based on the gray value of β-actin.
4
Western Blot Analysis of Adiponectin Signaling
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The cells were collected and the total proteins were extracted using an radioimmunoprecipitation assay (RIPA). Protein concentration was determined using a bicinchoninic acid protein assay. Equal amounts of protein (50 μg) were resolved on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Bedford, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline with Tween (TBST) and probed with antibodies against AdipoR1, p-AMPK, NF-κB-p65, and GAPDH (Cell Signaling Technology, Danvers, USA) at 4°C overnight. After 3 washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at 37°C. Immuno reactive bands were visualized using an enhanced chemiluminescence (ECL) detection system (Promega) and analyzed using Image-Pro Plus v. 6.0 software (Media Cybernetics, Inc., Rockville, USA).
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