Fig. 2.
Activated Ack1 binds and tyrosine phosphorylates AR in vivo and in vitro; primary human androgen-independent prostate cancer specimens express tyrosine-phosphorylated AR and Ack1. (A and B) LNCaP or LAPC-4 cells expressing myc-tagged Ack1 constructs were immunoprecipitated by using antibodies described, followed by immunoblotting analysis. (C) Schematic of GST-AR and GST-cAR constructs. TAD, transactivation domain; DBD, DNA-binding domain; LBD, ligand-binding domain. (D) Purified GST-AR and GST-cAR proteins were incubated with caAck or kdAck and ATP. Ack and GST-AR proteins were separated, electrophoresed, and subjected to immunoblotting analysis. (E) Equal amounts of protein from lysates of AICaP, ADCaP, and BPH samples were subjected to immunoprecipitation, followed by immunoblotting as indicated. (F) The levels of tyrosine-phosphorylated AR and Ack1 from immunoblots of AICaP samples were quantified by using SCION Image (Frederick, MD) software and plotted in arbitrary units. Pearson's product-moment correlation coefficient (r) and Spearman's rank correlation coefficient (rs) were calculated by using SAS statistical software, version 9.1 (SAS Institute, Cary, NC).