Fig. 8.
Intracellular Localization of GFP-Tagged PH Domain Mutants
HEK293 cells were grown on poly-l-lysine-coated cover-slips and transiently transfected with either pNEGFP, pNEGFP-IRS1-PH wt, pNEGFP-IRS1-PH S24A, or pNEGFP-IRS1-PH S24D. Monolayers were serum starved (overnight) 48 h post transfection, washed with PBS, fixed in 2% formalin, and mounted in aqueous mount. Confocal fluorescent images were captured using a Zeiss imaging microscope set to capture GFP and DAPI blue fluorescence from a slice thickness of 0.8 nm using a ×60 oil emersion objective. Representative pictures are shown from at least three independent experiments performed in duplicate.