Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters
Figure 2
Expression of epitope-tagged AUX1 in mammalian cells. (a) HEK293T cells were transfected using polyethyleneimine with the indicated amounts of recombinant L2-His63xFLAG or N-His63xFLAG epitope-tagged AUX1 DNA (L2 and N, resp., below each lane), and harvested 72 hours posttransfection. (b) HEK293T cells were transfected using 8 g of either L2-His63xFLAG-AUX1 or N-His63xFLAG-AUX1 epitope-tagged AUX1 DNA and harvested at the indicated times posttransfection. Cells were lysed by sonication, and 10 g of lysates were resolved by SDS-PAGE and identified by western blotting with anti-FLAG antibodies. The asterisk identifies a nonspecific protein reacting with the anti-FLAG antibody. Molecular weight (kDa) of marker proteins is denoted at the right-hand side of the figure. (c) HEK293T cells were transfected on coverslips in 6-well dishes with cDNA encoding N-His63XFLAG-AUX1 and were visualised 48 hours later by confocal microscopy after immunoblotting with an anti-FLAG primary antibody ( dilution) and a GFP-conjugated secondary antibody . Cell nuclei were counter stained with DAPI. (d) Transport of [3H]-IAA into U2OS cells transiently transfected with L2-His63XFLAG-AUX1 compared to transport into cells transfected with empty vector. Data is expressed as a percentage of the transport rate into AUX1-transfected cells and represents the mean ( standard error) of 9 independent experiments with 2–4 determinations of transport in each transfection.
