Samples were lysed in Laemmli buffer, separated by polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to an Immobilon polyvinylidene fluoride membrane (Millipore). Antibodies against adenosine monophosphate-activated protein kinase (AMPK, Cell Signaling, 2532S), phosphorylated AMPK (pAMPK, Cell Signaling, 2532S), Phosphorylated E1α (Cal Biochem, AP1062) and actin (Abcam, ab6276) were used.
Ap1062
Manufactured by Abcam
AP1062 is a lab equipment product. It is a device used in scientific research and experimentation. The core function of AP1062 is to perform specific tasks or measurements required in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab6276, by Abcam (1 mentions)
Anti mpc2, by Cell Signaling Technology (1 mentions)
Anti mcherry, by Cell Signaling Technology (1 mentions)
Tris glycine gel, by Bio-Rad (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Ab110025, by Abcam (1 mentions)
Ab168379, by Abcam (1 mentions)
Ab3280, by Abcam (1 mentions)
Ab68164, by Abcam (1 mentions)
3 protocols using ap1062
1
AMPK Activation Pathway Analysis
2
Western Blot Quantification Protocol
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Protein concentrations were determined using a Pierce MicroBCA kit (ThemoFisher), and 40 μg of protein was loaded into the lanes of precast criterion 4–15% tris-glycine gels (Bio-Rad) and electrophoresed. Proteins were then transferred to Immobilon PVDF membranes (MilliporeSigma). Membranes were blocked with 5% BSA in TBS-T for at least 1 h at room temperature. Primary antibodies were used at 1:1000 dilution in 5% BSA-TBS-T overnight; then, membranes were washed 3× in TBS-T and subjected to LiCor near-IR secondary antibodies at 1:10,000 dilution in 5% BSA-TBS-T for 1 h. After washing 3× in TBS-T, membranes were developed using a LiCor Odyssey imaging system. Antibodies used were anti-mCherry (Cell Signaling 43590S), anti-MPC2 (Cell Signaling 46141S), anti-phosphorylated PDH E1α S232, S293, and S300 (MilliporeSigma AP1062, AP1063, AP1064), anti-PDH Cocktail (Abcam ab110416), and anti-αTubulin (Sigma T5168); secondary antibodies used were anti-rabbit or anti-mouse IRDYE donkey 800CW (LiCor 926-32213 or 92632212). Full, un-cropped western blot images are provided in Supplemental Figure S1 .
3
Immunoblot Analysis of PDK and PDH
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Immunoblots were performed with SDS-polyacrylamide gel electrophoresis as previously described and analyzed using ImageJ (National Institutes of Health) (12, 25) . Antibodies used were as follows: PDK1 (Abcam, ab110025; detected at 49 kDa), PDK2 (Abcam, ab68164; detected at 46 kDa), PDH-E1a (Abcam, ab168379; detected at 43 kDa), phospho-Ser 293 -PDH-E1a (EMD Millipore, AP1062; detected at 44 kDa), and actin (Abcam, ab3280; detected at 42 kDa). The PDK-PDH antibodies have been validated in a recent publication (52) .
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