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Figure 1 | BMC Biochemistry

Figure 1

From: Beta-arrestin inhibits CAMKKbeta-dependent AMPK activation downstream of protease-activated-receptor-2

Figure 1

PAR 2 promotes AMPK activation in NIH3T3 cells. A-C. Representative westerns of phospho- and total AMPK (A, C) or phospho- and total Acetyl CoA Carboxylase (pACC) (B) in lysates from cells treated with or without 100nM 2fAP (to activate PAR2) for 0-120 minutes (A, B) or treated with a negative control peptide containing the reverse tethered ligand sequence, 2-Furoyl-LRGIL-O (RAP) (C). Integrated band intensity was determined for phospho- and total AMPK and Acc using LICOR Odyssey software and phosphorylated protein was normalized to total protein levels for each sample. Graphs of fold changes over baseline (D, F) or raw values for normalized phospho-protein (E, G) are shown for AMPK (D, E) or Acc (F, G). Baseline is defined as pAMPK levels observed in the absence of 2fAP treatment. AU refers to integrated intensity values as arbitrary units. Statistically significant differences between treated and baseline are indicated by ** (p < .005) and * (p < .05). G-H. To determine AMPK activity, AMPK was immunoprecipitated from cells after treatment with 2fAP for 0-120 minutes, incubated with the substrate SAMS peptide in the presence of 32P-ATP and reactions spotted onto phospho-cellulose filters. Radiolabel incorporation was determined by scintillation counting of filters and nmoles of phosphate incorporated per mg of AMPK was calculated (H). Fold changes in AMPK activity were determined as the ratio of radiolabel incorporated in treated over untreated controls (I). (*statistically significant increase in activity, p < .05, n = 8).

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