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Figure 8 | BMC Biochemistry

Figure 8

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

Figure 8

β-arrestin-2 inhibits CAMKKβ activity and AMPK phosphorylation in vitro. A, B. Conditions for optimal CAMKKβ concentrations and reaction times were determined. A. Increasing concentrations (0-100ng) of recombinant CAMKKβ were incubated with 1 μg of the substrate MBP in the presence of 32-P-ATP for 15 minutes. Reactions were analyzed by SDS-PAGE and radiolabel incorporation into excised MBP bands was determined. The upper panel is a representative autorad and the lower panel is the same gel stained with Coomassie Blue to show protein loading. B. Recombinant CAMKKβ (50ng) was incubated with 1 μg MBP and 32-P-ATP for 0-40 minutes and analyzed as described above. C-D. CAMKKβ kinase reactions using MBP as a substrate were repeated (50ng CAMKKβ, 15 minute reactions) in the presence of increasing concentrations of either recombinant GST or β-arrestin-GST and 32P-ATP. C. Representative autorad (Upper) of kinase reaction and corresponding coomassie stained gels (lower) are shown in the upper two panels. Levels of GST and β-arrestin-2-GST are shown in the inset at the bottom. D. Mean μ SEM CAMKKβ activity in the presence of GST and GST-β-arrestin-2 is shown (n = 4). E. Recombinant AMPK was incubated with or without CAMKKβ alone, and with CAMKKβ in the presence of 500pM GST or 500pM GST-β-arrestin-2. Samples were analyzed by SDS-PAGE followed by western blotting with anti-phospho-AMPK (upper) and anti-total AMPK (lower) F. Graphs depicting normalized pAMPK levels (left panel) and fold increase in pAMPK relative to baseline (right panel). Baseline is defined as AMPK phosphorylation observed in the absence of CAMKKβ, n = 3.

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