Fig. 2.

Translocation of green fluorescent protein (GFP)-labeled pleckstrin homology (PH) domains of general receptor for phosphoinositide-1 (GRP1) and protein kinase B (Akt) to the plasma membrane (PM) in response to acute stimulation with insulin. A: representative experiment. Fully differentiated 3T3-L1 adipocytes were electroporated with pPH-GRP1GFP as described in research design and methods. Later (∼24 h), they were preincubated in media containing 1% FBS with 5 or 25 mM glucose, with or without 0.6 nM insulin, for 18 h. They were then deprived of insulin and FBS for 2 h and either fixed in the basal state with 4% paraformaldehyde or stimulated with 100 nM insulin for 5 min for later analysis by confocal microscopy. At least 10 fields were analyzed from each dish, and each condition was tested in duplicate. Translocation was assessed by two “blinded” observers; the average scores assigned for each cell are shown. B: mean scores ± SE from 6 separate experiments in which the translocation of PH-GRP1GFP was tested. The conditions of preincubation are indicated below the bars; acute insulin stimulation (basal vs. 100 nM insulin) are indicated by the colors of the bars. C: mean scores ± SE from 4 experiments in which the translocation of PH-AktGFP was tested in response to acute stimulation with 0.6 or 100 nM insulin. The conditions of preincubation are indicated below the bars.