Fig. 1. Serum starvation promotes tyrosine phosphorylation of a 145 kDa protein in a Src- and EGFR-dependent manner in bladder carcinoma 5637 cells. (A) Human 5637 cells (1x10⁶ cells/dish) were cultured in normal conditions (10% FCS) for 48 hours. After the treatment, cells were cultured for an additional 48 hours in normal conditions (), 10% FCS plus 10 µM PP2 (), without serum () or without serum plus 10 µM PP2 (). Cell number was determined at 24, 48, 72 and 96 hours post initial treatment. (B) Whole cell extracts (30 µg/lane) were prepared from carcinoma 5637 cells that had been cultured in normal (10% FCS, denoted FCS) or serum-free medium (for 1 to 48 hours), and analyzed by immunoblotting (IB) with an anti-phosphotyrosine antibody (PY99), an antibody against the activated and tyrosine-phosphorylated Src family protein-tyrosine kinases (pSFK) (pY418) or an anti-ß tubulin antibody. In the top panel (IB: PY99), the positions of a 145 kDa tyrosine-phosphorylated protein (pp145) and molecular size markers (25-250 kDa) are indicated. (C) 5637 cells were cultured in normal (+ FCS) or serum-free (– FCS) medium for 24 hours. For the final 1 hour of the serum starvation treatment, cells were incubated in the absence or the presence of the following substances: 50 µM genistein (Gen), 50 µM daidzein (Ddz), 10 µM PP2, 10 µM PP3, 1 µM SU6656 or 5 µM AG99. Whole cell extracts (30 µg/lane) were analyzed by immunoblotting with anti-phosphotyrosine antibody. The position of pp145 is indicated. (D) Carcinoma 5637 cells were treated as in panel A. After the treatments (96 hours post treatment), cell death (black bars) and caspase 3/7 protease activity (grey bars) of the whole cell extracts (20 µg/assay) were determined by Trypan Blue exclusion and a synthetic substrate Ac-DEVD-AMC, respectively. Data shown are mean ± s.d. of three independent experiments. *P<0.01 compared with control.