FIGURE 2.

BacMam baculovirus-transduced HeLa cells exhibit altered cell surface expression and transport function of single (D164C and D805C) and double (D164C/D805C) mutant P-gp at 37 °C. P-gp-virus-transduced HeLa cells were incubated with UIC2 antibody (2 μg/100,000 cells) for 30 min at 37 °C followed by incubation with FITC-conjugated anti-mouse IgG2a secondary antibody. The detection with UIC2 was carried out both in the absence and presence of 20 μm CsA, as described in the UIC2 shift assay under “Experimental Procedures.” The cell surface localization of cysless-WT along with the three mutant P-gps (D164C, D805C, and D164C/D805C), as detected by UIC2 antibody, is shown in panels A–C, respectively. The blue and green histograms show the UIC2 detection of cysless-WT and mutant-Pgp, respectively, in the absence of CsA, whereas the orange and red histograms mark the UIC2 shift upon CsA treatment for cysless-WT and mutant P-gps. Panels D–F show typical histograms for accumulation of daunorubicin in HeLa cells expressing D164C, D805C, or D164C/D805C. The transport function of the three mutant P-gps was compared with cysless-WT (blue, positive control) and E556Q/E1201Q (referred to as EQ)-mutant P-gp (red, negative control), which is known to be completely functionless (22). The quantification of data and number of experiments are given in Table 1.