Figure 1

Characterisation of the specificity of the ISET/immunohistochemistry methodology for the androgen receptor utilising cells lines as controls. Specificity was investigated by employing human prostate cancer cell lines of known AR status, together with treatment of cells with AZD3514 in order to modulate the levels of the protein, to spike volunteer blood samples prior to processing by ISET and analysis by IHC. Untreated LNCaP cells were the positive control and demonstrated strong brown nuclear staining for AR. PC3 cells were the negative control and displayed a complete absence of brown staining. LNCaP cells pre-treated for 24 hours with 10 μM AZD3514 prior to spiking into blood demonstrated a marked reduction in the level of nuclear staining, compared to untreated LNCaP cells. Inset: Western blot analysis of LNCaP and PC3 cells for androgen receptor protein expression. Loading was normalised by the addition of 20 μg of protein to each lane and the blot was run according the standard western blot protocols, utilising anti-androgen receptor antibody clone AR441 as the primary antibody and enhanced chemiluminescence detection for visualisation of bands.