Figure 5.
SET9 enhances AR N- and C-terminal interaction without affecting receptor stability or nuclear shuttling. (A) HEK293T cells transiently transfected with empty vector, SET9 or SET9H297A were treated with 1 µM cycloheximide (CHX) for up to 8 h and then subject to western analysis using anti-AR and anti-α-tubulin antibodies. (B) LNCaP cells transiently transfected with non-silencing (N/S) or SET9 siRNAs were treated with 1 µM CHX and subject to western analysis as in (A). (C) LNCaP cells transiently transfected with N/S or SET9 siRNAs in steroid-depleted media were treated with and without 10 nM DHT for 6 h prior to nuclear-cytoplasmic extraction and western analysis using anti-AR, -PARP1 and -GAPDH antibodies. (D) HEK293T cells transiently transfected with AR or ARK632R were subject to the same experimental procedure as in (C) with the inclusion of an additional 10 nM DHT time-point at 2 h. (E) Mammalian two-hybrid analysis of AR-TD and AR-DBD/H/LBD fragment interaction in HEK293T cells transiently transfected with either SET9, SET9H297A or SRC-1 and Gal4-luciferase and β-galactosidase reporters. Cells were treated with and without 10 nM DHT for 24-h prior to harvesting, and luciferase and β-galactosidase analyses. Data represents the average of three independent experiments performed in quadruplicate ± standard error (asterisk represents statistical significance <0.05).