Fig. 3.

Increased glucose uptake by PI 4,5-P2 replenishment after PBP10 treatment is not attributable to recruitment of GLUT4 or GLUT1. 3T3-L1 adipocytes (A, C) or 3T3-L1 adipocytes expressing HA-GLUT4-EGFP (B) were infected either with control LacZ adenovirus (control) or with recombinant adenovirus for expressing human type 1α PIP-kinase (PIPK). Cells were then either left untreated or treated with 100 nM insulin or with 40 μM PBP10 for 15 min. For washing out PBP10, cells were, then, washed with the medium and incubated in the medium for 20 min. (A) Membrane sheets were prepared as described in Materials and methods. A representative image from three independent experiments is shown. For quantifying the amount of GLUT4 in the membrane sheets, the average values±S.E. obtained from three independent experiments are shown. N.S., not significant. (B) Cells were immunostained for the HA-tag without permeation and the amount of the HA-tag exposed at the cell surface was quantified as described in Materials and methods. The amount of the HA-tag on the cell surface per the amount of EGFP expressed in each cell was measured. The average value±S.E. obtained from three independent experiments, in which at least 20 cells were counted, are shown. N.S., not significant. (C) Membrane sheets were prepared and stained for GLUT1. A representative image out of three independent experiments is shown.