Figure 3. The mTOR/translational pathway
was
involved in IFNγ-induced VEGF secretion from RPE cells. A:
ARPE-19 cells were cultured with or without IFNγ in the presence or
absence of rapamycin for 15, 30, and 60 min. Cells were collected and
processed for western blot analysis using anti-p-p70 S6 kinase,
anti-p-S6 ribosomal protein, anti-p-4E-BP1, p-akt, and GAPDH
antibodies. B: ARPE-19 cells were cultured with or without
IFNγ/rapamycin for 24 h. Cells were collected for RNA purification.
Real-time PCR assay was performed and the results were expressed as the
n-fold expression of hVEGF normalized on that of GAPDH or 18S rRNA. C:
ARPE-19
cells
were cultured with or without IFNγ/rapamycin for 48 h.
Cell supernatants were collected and used for ELISA analysis. The
values are expressed as the average+SEM of triplicates of each
treatment. The results were representative data from three separate
experiments. The double asterisk indicates statistical significance
(p<0.01) compared to the IFNγ group. D: ARPE-19 cells were
cultured with or without IFNγ in the presence or absence of rapamycin
for 15, 30, and 60 min. Cells were collected and processed for western
blot analysis using anti-p-Stat1, Stat1, and GAPDH antibodies. The
results were representative data from two separate experiments.