FIGURE 3.

Cell differentiation causes redistribution of IRAP from heavy donor membranes into the GSVs. A, undifferentiated (Fb) and differentiated (Ad) 3T3-L1 cells stably expressing IRAP-EYFP were homogenized and centrifuged at 16,000 × g for 20 min. Supernatants (S, 100 μg/lane) and pellets (P, 100 μg/lane) were separated by PAGE. The panel shows Ponso Red-stained gel. B, individual proteins were analyzed in supernatants (S) and pellets (P) by Western blotting with specific antibodies. C, supernatant obtained from differentiated cells as explained in the legend to panel A was centrifuged in a 10-30% linear sucrose gradient for 65 min in a Sorvall TST60.4 rotor at 48,000 rpm. Gradient fractions, including the pellet of this centrifugation (P), were analyzed by Western blotting. The arrow indicates the direction of sedimentation. D, vesicle reconstitution assay was performed in vitro in duplicate with donor membranes and cytosol isolated from differentiated and undifferentiated wild type 3T3-L1 cells. The lower panel shows the quantification of the Western blot stained with anti-IRAP antibody. The absence of the error signs on the bars indicates that the error is virtually undetectable. A representative result of three independent experiments is shown. a.u., arbitrary units. E, differentiating 3T3-L1 cells were separated into 16,000 × g supernatant and pellet on each day of differentiation as explained in the legend to panel A. Endogenous IRAP in the supernatant (sup) and pellet was analyzed by Western blotting and expressed as the ratios (mean values ± S.E. of three independent experiments) between IRAP content in these fractions on each day of differentiation.