Figure 4.

Effect of PF-4989216 on photoaffinity labeling of ABCG2 with [¹²⁵I]IAAP and protein expression of ABCG2. (A) Membrane vesicles (500 μg protein/mL) from MCF7-FLV1000 cells expressing ABCG2 was incubated in the presence or absence of 10 μM of PF-4989216 at 21 °C in 50 μmol/L Tris-HCL (pH 7.5) and 150 mM NaCl. [¹²⁵I]IAAP (2,200 Ci/mmol) at 3 to 6 nmol/L was added to the samples and incubated for 5 min under subdued light before illuminated with an UV lamp (365 nm) for 10 min at room temperature. The labeled ABCG2 was immunoprecipitated with BXP-21 antibody and visualized as described previously.³¹ (B) Immunoblot detection of human ABCG2 (upper panel) and quantification (lower panel) of total lysate protein (10 μg) from drug-sensitive S1 and drug-resistant S1-M1–80 cells treated with increasing concentrations (0–3 μM) of PF-4989216 for 72 h as indicated. α-Tubulin was used as loading control. Representative autoradiogram and values represent mean ± SD calculated from three independent experiments.