FIGURE 2.

Analysis of the abundance of 14-3-3 isoforms in rat adipocytes and their interactions with AS160. A, quantitative real-time PCR analysis of the relative abundance of mRNA for the different known 14-3-3 isoforms in rat adipocytes. B, quantification of the 14-3-3β, -γ, and -ϵ proteins in rat adipocytes as detected by immunoblotting and quantified by comparison with a recombinant protein standard curve as illustrated in Fig. 4. C, comparison of the relative affinity of recombinant 14-3-3 isoforms β, γ, and ϵ for AS160. Equal amounts of GST-14-3-3β, -γ, or -ϵ immobilized on glutathione beads were incubated with total cell lysate prepared from basal (B) or insulin (I)-stimulated rat adipocytes. Relative amounts of AS160 pulled down by the recombinant proteins were detected by immunoblotting with anti-AS160 antibody. Results are means ± S.E. from three independent experiments and with a representative immunoblot (D). E, comparison of the interaction of recombinant 14-3-3 isoforms β, γ, and ϵ with phosphorylated AS160. Equal amounts of GST-14-3-3β, -γ, or -ϵ immobilized on glutathione beads were incubated with cell lysates prepared from basal or insulin-stimulated rat adipocytes. Relative amounts of phosphorylated AS160 pulled down by the recombinant proteins were detected by immunoblotting with anti-PAS antibody. Results are means ± S.E. from three independent experiments with a representative immunoblot (F).