(A) shows RT-PCR analysis of the expression of EFR3A and EFR3B normalized to GAPDH in 3T3-L1 fibroblasts and adipocytes. Data presented is from triplicate biological repeats each with at least three technical replicates (mean and S.D.) EFR3A is the predominant isoform in 3T3-L1 fibroblasts and adipocytes. (B) shows a subcellular analysis of 3T3-L1 adipocytes treated with or without 100 nM insulin for 20 min and separated into PM (PM)-enriched, high density microsomes (HDM) and low density microsomes (LDM), which were then immunoblotted for the proteins indicated; figures at right are approximate positions of MW markers in kDa. In each case, 30 μg of protein was loaded in each fraction. Data from a representative experiment is shown, replicated four times with qualitatively similar results. (C–E) shows quantification of GLUT4 (C), EFR3 (D) and PI4K-IIIα (E) signals in PM and LDM fractions in the four fraction experiments. GLUT4 levels in the PM increase 1.7-fold (C, *P=0.002) and decrease in the LDM by 25% (**P=0.003) consistent with similar studies. EFR3 and PI4K-IIIα levels in the PM-enriched fractions increase in response to insulin (D, 2.1-fold, ***P=0.012 and E, 1.9-fold, ****P=0.03, respectively). Modest decreases in the LDM fraction for these proteins do not reach statistical significance (n.s.). Syntaxin4 (Sx4) is used as a marker for a protein known to be enriched in the PM [55,56].