Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters
Figure 3
Expression of epitope-tagged AUX1 in insect cells. (a) Sf9 cells at densities of cells/mL were infected with recombinant baculoviruses expressing epitope-tagged AUX1 at a range of multiplicity of infections (MOIs) and harvested at 24–96 hours postinfection (hpi). Cell lysates (10 g) were resolved by SDS-PAGE and identified by western blotting with appropriate antibodies directed towards the epitope tags. Panels show data for a 72-hour postinfection only. Molecular weight (kDa) of marker proteins is denoted at the right-hand side of the figure. (b) Auxin binding to AUX1-containing membranes (72 hours postinfection) was assessed by a centrifugation-based radioisotope binding assay [12]. AUX1 displacement could be observed when membranes were incubated with 1 mM auxin analogues such as 2,4-D and 1-NOA but not when the unrelated weak acid benzoic acid (BA) was applied. (c) Solubilisation of HA-AUX1 from insect cell membranes. 100 g of membranes were incubated for 60 minutes at C with detergents at greater than 2X critical micelle concentration. Solubilised material was separated from insoluble material by ultracentrifugation and equal percentages of the two fractions resolved by SDS-PAGE and immunoblotting. Detergent abbreviations: 3–16: Zwittergent 3–16 (Calbiochem); -OG: -octyl-glucoside; DM: n-decyl--D-maltoside decylmaltoside; DDM: n-dodecyl--D-maltoside.
