Figure 2. HOTAIR Directly Interacts with the AR Protein.

(A) Western blot showing AR protein interaction with HOTAIR RNA. RNA pull-down assay was performed in LNCaP and C4-2B cells using biotin-labeled HOTAIR RNA probe transcribed in vitro from the first 360 bp of the sense HOTAIR gene and detected using western blots. No probe and HOTAIR 1–360AS probe were used as negative controls.

(B and C) The qRT-PCR showing HOTAIR enrichment by an anti-AR antibody. LNCaP (B) and C4-2B (C) cells were subjected to RIP assay using an anti-AR antibody or IgG. IP-enriched RNA was then analyzed by qRT-PCR using U1 RNA as a negative control. (Bottom) Western blots confirm AR protein IP. Error bars are mean ± SEM.

(D) The 5′ end of HOTAIR transcript binds AR. RNA pull-down was carried out in LNCaP cells with various HOTAIR deletion probes (top), and the retrieved protein extract was analyzed by western blotting (bottom).

(E) NTD of the AR protein interacts with HOTAIR. FLAG-tagged AR deletion constructs were transfected into 293T cells and the expression confirmed by western blot analysis (left). RNA pull-down using biotinylated HOTAIR 1–360S probe retrieved the NTD-containing AR proteins (right).

(F and G) Coomassie blue staining verifying purified recombinant MBP-AR-NT protein synthesized in vitro (F) and western blot of AR-NT proteins retrieved by increasing doses of HOTAIR probes in in vitro binding assay (G). HOTAIR 1–360AS probe was used as a negative control.