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. Author manuscript; available in PMC: 2014 Mar 14.
Published in final edited form as: Prostate. 2010 Sep 1;70(12):1274–1285. doi: 10.1002/pros.21163

Fig. 3.

Fig. 3

Ack1 targeted ARTyr267-phosphorylation is resistant to anti-androgens. A: Serum and androgen-depleted LNCaP cells were treated with heregulin (10 ng/ml, 45 min) and bicalutamide (1 μM, 16 hr) or flutamide (10 μM for 16 hr). Cell lysates were immunoprecipitated using pTyr267-AR antibodies, followed by immunoblotting with AR antibodies (top panel). B: Serum and androgen-depleted LAPC4 cells were treated with EGF (10 ng/ml, 40 min) and bicalutamide (1 μM, 16 hr) or flutamide (10 μM for 16 hr). Cell lysates were immunoprecipitated with pTyr-antibodies, followed by immunoblotting with pTyr267-AR antibodies (top panel). C: HEK293 cells were transfected with the ARR2PB-luciferase reporter (500 ng) and FLAG-tagged AR vector (500 ng). Twenty-four hours after transfection, cells were treated with DHT (2.5 nM, 16hr) and bicalutamide (1 μM, 16 hr) or flutamide (10 μM, 16 hr) and luciferase activity was determined. Equal expression of AR was confirmed by immunoblotting the lysates with anti-FLAG antibodies. Data are representative of three independent experiments. *P≤0.001; **P≤0.04. D: HEK293 cells were transfected with the ARR2PB-luciferase reporter (250 ng), AR vector (250 ng), and caAck1 (250 ng). Twenty-four hours after transfection, cells were treated with bicalutamide (1 μM, 16 hr) or flutamide (10 μM, 16 hr), and luciferase activity was determined. Equal expression of AR was confirmed by immunoblotting the lysates with anti-FLAG antibodies. Data are representative of three independent experiments. E: LAPC4 cells were transfected with ARR2PB-luciferase reporter construct, serum and androgen-depleted, treated with EGF (10 ng/ml) for various time points and luciferase activity was determined. F: LAPC4 cells were transfected with ARR2PB-luciferase reporter construct, serum and androgen-depleted, treated with DHT (2.5 nM, 16 hr), bicalutamide (1 μM, 16 hr), or flutamide (10 μM, 16 hr), and luciferase activity was determined. G: LAPC4 cells were transfected with ARR2PB-luciferase reporter construct, serum and androgen-depleted, treated with EGF (10 ng/ml, 4 hr), bicalutamide (1 μM, 16 hr), or flutamide (10 μM, 16 hr), and luciferase activity was determined.