FIG. 5.
Dominant-negative Etk (A) and Src (C) inhibits bombesin-mediated cell growth using the BrdU labeling proliferation assay. (A) LNCaP-pcDNA3, LNCaP-EtkWT, and LNCaP-EtkDN cells were incubated in the absence (bar 1) or presence of different concentrations of bombesin as follows: 0.1 nM (bar 2), 1 nM (bar 3), 100 nM (bar 4), and 1 μM (bar 5) in 10% charcoal-stripped FBS for 72 h. At the end of incubation, cells were fixed and stained with BrdU as specified by the manufacturer's protocol and fold increase was measured by ELISA. Each experiment was carried out in triplicate, and the error bars represent standard deviations. For each bar, the fold increase was normalized to the value for the control group. (B) LNCaP cells were not treated (lane C) or treated with different concentrations (nanomolar) of bombesin (Bomb) as indicated for 30 min. Tyrosine phosphorylation of Etk was analyzed by immunoprecipitation using anti-Etk antibody (IP:αEtk) followed by Western blotting (immunoblotting) with anti-pY antibody (IB:αp-Y). Anti-phospho-tyrosine (top blot) (αEtk) (active Etk) and anti-T7 (bottom blot) (αT7) (total Etk) antibodies were used in Western blots (immunoblots [IB]) of Etk immunoprecipitates. Numbers under the bands indicate the fold activation of Etk, as quantitated by video image densitometry. (C) LNCaP cells were preincubated in the absence or presence of PP2 (10 μM) for 30 min before the addition of R1881 (1 nM), or bombesin (Bomb) (100 nM) for 72 h under charcoal-stripped serum conditions. Then 20 μl of MTS was added to each well for 2 h at 37°C. After incubation, the absorbance or optical density at a wavelength of 490 nm (OD490nm) was read.