Figure 4.

PHB:E2F1 interaction is inhibited by androgen treatment. (a) Western blot of PHB levels from PHB, AR and E2F1 immunoprecipitates from nuclear extracts from LNCaP cells hormonally starved for 72 h followed by treatment with 10 nM R1881 for 4 h. (b) Abrogation of PHB:E2F1 co-immunoprecipitation when extracts were preincubated with alkaline phosphatase enzyme. (c) Upper panel—schematic diagram of the Checkmate two-hybrid interaction assay for PHB and E2F1. Lower panel—luciferase activity from LNCaP cell extracts transfected with components of the checkmate assay including pBIND^Gal4-PHB and pACT ^VP16-E2F1, along with empty vector controls. Transfections were normalised to β-galactosidase. (d) Upper panel—schematic diagram of the NanoBit interaction assay for PHB and E2F1. Lower panel—nano-luciferase activity in live LNCaP cells grown in charcoal-stripped serum and transfected with components of the NanoBit assay including pPHB-LargeBit and pE2F1-SmallBit, along with empty vector controls (Halo-tagged Large+SmallBit), positive interaction controls (PRKACA:PRKAR2A) and positive rapamycin-inducible interaction control (FRB:FKBP). Cells were then treated with androgen and measured again at 2 h. All data are the mean±s.d. of three independent experiments performed in triplicate. *P<0.05 and **P<0.01 (t-test analysis).