FIG 6.

WT GIPR and E354Q GIPR recycle back to the cell surface after GIP challenge. (A and B) Adipocytes were treated without or with 100 nM GIP for 1 h, washed and fixed, or washed and further incubated in serum-free medium for the times indicated. Plasma membrane GIPR (A) and total GIPR (B) levels were determined. Each graph represents averages ± standard errors of the means of data from 3 to 4 independent experiments (**, P < 0.01; *, P < 0.05). (C and D) Epifluorescence images of adipocytes expressing WT or E354Q HA-GIPR-GFP, stained for TGN46. Cells were incubated without or with 100 nM GIP for 1 h. One set of GIP-treated cells was washed and incubated in serum-free medium for an additional 1 h. The cells were fixed, permeabilized, and stained with anti-TGN46 antibodies, followed by secondary staining with anti-rabbit Cy3 antibodies. Bar, 10 μm. (E) Confocal optical sections of WT HA-GIPR-GFP and E354Q HA-GIPR-GFP colocalization with TGN46 in adipocytes. Arrows indicate colocalization of GIPR with TGN46 in the perinuclear region of cells.