Figure 5

Results of western analysis support the rank order of DAT density observed in saturation binding experiments. A) Lane 1: molecular weight marker, Lane 2: negative control, Lane 3: hDAT transfected cell line, Lane 4: hDAT Zero transfected cell line, Lane 5: hDAT 9 transfected cell line, Lane 6: hDAT 10 transfected cell line. Blots were probed both with a rat anti-DAT monoclonal antibody specific for the transporter's N-terminal region as well as a 14-3-3β polyclonal antibody to control for possible protein loading differences. DAT immunoreactivity was detected with an Alexa Fluor 680 conjugated anti-rat IgG (red) and 14-3-3β immunoreactivity with an IRDye-800 conjugated anti-rabbit IgG (green). B) DAT immunoreactivity was normalized to the 14-3-3β signal, densiometrically quantified with respect 14-3-3β content, and analyzed via a one way ANOVA with a Holm-Sidak post-hoc analysis. Statistically significant differences (* = p < 0.05) were detected for all comparisons except the hDAT10-vs-hDAT Zero comparison.