Fig 1: Expression of AMPKβ isoforms in different adipocyte models. Cell lysates were prepared from 3T3–L1 adipocytes or adipocytes isolated from mouse or rat epididymal- or human subcutaneous adipose tissue. (A–E) AMPKβ1 or β2 was immunoprecipitated (IP) from lysates using isoform-specific antibodies. Whole lysates ( = lysates prior to IP), IPs, and supernatants collected after IP (immuno-supernatants, IS), were analyzed by western Western blotting (A). AMPKβ1 and β2 signals in whole lysates (B) or in immuno-supernatants (IS) collected after β1- (C) or β2 (D) IP were quantified and expressed as β1/β2 (B) or % of the corresponding signal from the whole lysate (C, D). (E) Quantified AMPKα signals from immuno-supernatants (IS) collected after β1- or β2 IP were expressed as % of AMPKα in the whole lysate. (F) AMPK in vitro kinase activity was determined after α1- (black bar), β1- (left white bar) or β2 IP (right white bar). (G) AMPKα1 in vitro kinase activity was determined in immuno-supernatants (IS) collected after β1- or β2 IP in F. (F, G) Data is expressed as % of AMPKα1 activity in the lysate and absolute activity (mU/mg) is shown in Supplementary Figure S1. (H) Ratio of AMPKβ1 and β2-associated in vitro kinase activity determined in Supplementary Figure S1A. All data (A–H) is expressed as mean of three independent experiments ± SD. (I) AMPKβ1 and β2 in lysates of adipocytes isolated from mouse epididymal (epi) and inguinal (ing) adipose tissue from the same mouse (n = 3) were analyzed by western blotting (left panel) and quantified signals were expressed as β1/β2 (average epi = 1.26, average ing = 1.79; right panel). Statistical significance was determined by unpaired (E–G) or paired (I), two-tailed, Students t-test * p 0.05, **pp 0.01, ns = non–significant.