Figure 7. Induction of rab11 mRNA in Response to Estrogen in
Ishikawa Cells
Ishikawa cells were cultured and transiently transfected using
the lipofectamine procedure as described in Materials and
Methods. Experiments were performed with medium containing
charcoal-stripped serum. Semiconfluent cells received either empty
vector (lanes 1 and 2) or expression vectors containing ER
(lanes
3–6) or ERß (lanes 7 and 8). After 24 h, the cells were
incubated in fresh medium with ligands (10-7 M
estrogen or estrogen along with 10-5 M ICI
182,780) or solvent. Twenty four hours after treatment with ligands,
cells were harvested and mRNA was isolated and subjected to RT-PCR
analysis employing rab11 or GAPDH-specific primers. PCR reactions using
both rab11 and GAPDH primers were performed through multiple rounds
ranging from 15–40 cycles to ensure that the amplifications were in
the linear range. The data shown above represent 25 cycles of PCR
amplification. The authenticity of the PCR products was confirmed
by Southern blotting employing 32P-labeled rab11 and
GAPDH cDNA probes. Lanes 1, 3, and 7 represent cells treated with
vehicle; lanes 2, 4, and 8 represent cells treated with estrogen;
lane 6 represents cells treated with ICI 182,780; and lane 5 represents
cells treated with estrogen in combination with ICI 182,780. The
relative levels of rab11 mRNA were as follows: lane 1 (13 ±
1.75), lane 2 (12 ± 2), lane 3 (13 ± 1.25), lane 4
(100 ± 8), lane 5 (22 ± 2.5), lane 6 (11 ± 2), lane 7
(15 ± 1.5), and lane 8 (90 ± 5).
