Figure 7

Co-immunoprecipitation of β-arrestin-2, CAMKKβ and AMPK in NIH3T3 cells and primary fat tissue. A-C. NIH3T3 cells, transfected with empty vector (as a negative control for Co-IPs) or flag-tagged β-arrestin-1 or 2, were treated with 2fAP for 0-120 minutes, lysed and β-arrestins immunoprecipitated with anti-flag, followed by western blotting with anti-AMPK, CAMKKβ and Flag. Expression of β-arrestin-1 was higher than that of β-arrestin-2 so AMPK and CAMKKβ protein levels in each immunoprecipitation were normalized to normalized to β-arrestin-1 or 2 levels. Bar graphs depicting quantification of immunoprecipitated AMPK (B) and CAMKKβ (C) are shown (n = 4). D. Epidydimal fat from wt mice was harvested and lysates were immunoprecipitated with AMPK or IgG followed by western blotting with antibody to AMPK (upper), CAMKKβ (middle panel) and β-arrestin-1/2 (lower panel). IgG heavy chain can be seen at 50kD in the β-arrestin panel. The band above IgG corresponds to β-arrestin-1 and the band below corresponds to β-arrestin-1.