Protein lysates were prepared from frozen powdered liver using T-PER buffer (ThermoFisher #78510) containing protease inhibitor cocktail and phosphatase inhibitors from Sigma-Aldrich (St. Louis, MO). Western blot analyses were performed using standard PAGE-SDS as previously described (25 (link)). Primary antibodies were as follows: ACC (#3676), p-ACC (#11818), AMPKα (#5831), p-AMPKα (#2535), AMPKβ½ (#4150), p-AMPKβ1 (#4181), and Pex5 (#83020) from Cell Signaling; Catalase (ab16731), Total OXPHOS cocktail (ab110413), Pex19 (ab137072), and PMP70 (ab3421) from Abcam; GLUT2 (07–1402) and Pex14 (ABC-142) from EMD Millipore, and PGC-1α (sc-517380) from Santa Cruz Biotechnology. HRP-linked secondary antibodies (anti-mouse IgG Cat# NXA931 and anti-rabbit IgG Cat# NA934V) were purchased from GE Healthcare (Piscataway, NJ) and proteins were detected using ECL chemistry. Reversible protein stain-MemCode (MemC) (Thermo-Fisher #24580) was used to confirm equal transfer of proteins and quantitation of these bands served as a loading control. A ChemiDoc imaging system (Bio-Rad) was used to image bands and Image Lab software (Bio-Rad) was used to quantitate band intensity. Data are expressed as fold change compared to the Con-Sed group after normalizing to MemC.
P ampkβ1
Manufactured by Cell Signaling Technology
P-AMPKβ1 is a primary antibody that specifically recognizes the phosphorylated form of the AMPK beta 1 subunit. AMPK is a key energy sensor in cells that is activated in response to cellular stresses that deplete ATP levels.
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Lab products found in correlation
T per buffer, by Thermo Fisher Scientific (1 mentions)
Total oxphos cocktail, by Abcam (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
P acc, by Cell Signaling Technology (1 mentions)
Pgc 1α, by Santa Cruz Biotechnology (1 mentions)
Glut2 07 1402 and pex14 abc 142, by Merck Group (1 mentions)
Reversible protein stain memcode memc, by Thermo Fisher Scientific (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Lc3b 1, by Cell Signaling Technology (1 mentions)
Lc3b 2, by Cell Signaling Technology (1 mentions)
A 769662, by LC Laboratories (1 mentions)
2 protocols using p ampkβ1
1
Western Blot Analysis of Liver Proteins
2
AMPK Activation and Autophagy Regulation
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The AMPK activator A769662 was obtained from LC Laboratories (Woburn, Mass). The primary antibodies directed at AMPKα1/2, pAMPKα1/2, AMPK β1/2, pAMPKβ1, LC3B-I, and LC3B-II were obtained from Cell Signaling (Beverly, Mass); the primary antibody directed at PGC-1α, was obtained from Abcam (Cambridge, Mass). The primary antibodies directed at SIRT1 and β-actin and the secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). The Odyssey blocking and loading buffers and LI-COR secondary antibodies were obtained from LI-COR Biotechnology (Lincoln, Nebr). All other chemicals were obtained from Sigma-Aldrich (St. Louis, Mo).
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