Fig. 1. Rabip4 expression induces an increase in glucose uptake and Glut 4 translocation to the plasma membrane. 3T3-L1 Rabip4 Tet-off adipocytes were cultivated for 3 days with or without tetracycline (+/-tetra) to induce the expression of Rabip4. Cells were serum starved overnight, and then treated with insulin for 20 minutes. (A) Deoxyglucose uptake was then determined as described in the Materials and Methods. The results were expressed as the mean ± s.e.m. of five experiments of the fold increase in insulin stimulation over the basal condition of cells cultivated with tetracycline (no Rabip4 expression). (B) 40 µg protein from 3T3-L1 Rabip4 Tet-off adipocytes cultivated with or without tetracycline for 3 days were separated by SDS-PAGE and analyzed by western blot for the presence of Rabip4, Glut 4 and Rab4. (C) Glut 4 translocation was determined by the amount of Glut 4 present in plasma membrane sheet preparations as described in the Materials and Methods. Data are the mean ± s.e.m. of three experiments of the fold increase in insulin stimulation over the basal condition of cells cultivated with tetracycline (no Rabip4 expression). (D) 3T3-L1 Rabip4 Tet-off adipocytes cultivated with or without tetracycline for 3 days were serum starved overnight before being stimulated with 100 nM insulin. Insulin-induced tyrosine phosphorylation was detected using anti-phosphotyrosine antibodies. YP-IR, tyrosine-phosphorylated ß subunits of the insulin receptor; YP-IRS, tyrosine-phosphorylated insulin receptor substrates. Activation of protein kinase B (PKB) and extracellular-regulated kinases (ERK) was estimated using anti phospho-PKB and anti phospho-ERK. ^*P<0.001 compared with levels in the +tetra control.