Figure 2

Results from TSR analysis are valid across a broad range of carrier expression levels E. coli strains SK11 (GabP-positive) and SK45 (GabP-negative) were grown to early logarithmic phase as described in Methods except that expression was induced by exposing cultures to the indicated IPTG concentrations. The cells were washed with 100 mM potassium phosphate buffer (pH 7.0), and dual-label competitive transport reactions were initiated by exposing the cells to 7 μM [³H]NA (0.42 μCi/ml) and 3 μM [¹⁴C]GABA (0.06 μCi/ml) for 10 seconds (initial rate) at 30°C. Error bars represent the S.E.M. (n = 3). Panel A. GabP-dependent uptake (SK11 signal minus SK45 signal) of either [³H]NA (■) or [¹⁴C]GABA (▲). Panel B. Transport Specificity Ratio (GABA/NA). Inset. Immunoblot of plasma membrane vesicle protein (2 μg per lane) probed with an anti-pentaHis mAb and developed with a chemiluminiscent alkaline phosphatase substrate (see Methods). Lane 1: Membranes from E. coli strain SK45 (GabP-negative). Lanes 2–10: Membranes from E. coli SK11 (GabP-positive) grown in the presence of 2, 5, 10, 20, 50, 100, 200, 500, or 1000 μM IPTG, respectively.