Fig. 4.

Histogram of Tfn uptake in GAK-knockout MEF cells transfected with different constructs. (A) Internalization of AlexaFluor647–Tfn was measured in GAK-knockout cells that had been transfected with GFP, different GFP-labeled GAK fragments or GFP-labeled chimeras. Control (Con) cells, the MEFs derived from the GAK-knockout mice that still expressed endogenous GAK, were transfected with GFP. Cells were sorted based on the intensity of GFP and AlexaFluor647 fluorescence. From the fluorescence-activated cell sorting (FACS) plot, the median fluorescence intensity of AlexaFluor674 for each population of GFP-expressing cells was calculated. Data are from four independent experiments (mean±s.d.). GAK-FL, full-length GAK. (B) Internalization of Tfn in GAK-knockout cells that had been stably transfected with different GAK fragments. After establishing the stable cell lines, GAK was knocked out by treating cells with adenovirus expressing Cre recombinase. Control MEFs were the stable MEF cell lines derived from the GAK-knockout mice that still expressed endogenous GAK. In control cells and in the adenovirus-treated stable cell lines, AlexaFluor488–Tfn was internalized for either 5 min (open bars) or 10 min (shaded bars). Cells were sorted based on the intensity of the AlexaFluor488 fluorescence to determine the internalization of Tfn. Data are from three independent experiments (mean±s.d.).