LncRNA-HIT recruits a complex of p100 and CBP in the limb.

(A-D)Snd1 which encodes p100 is expressed in many of the same regions as LncRNA-HIT including the limbs at E11-13.5, spinal cord, and genital tubercle. FL = forelimb, HL = hindlimb, MX = maxillary component of the first branchial arch, SC = spinal cord, P = pinnae. (E) Western blot analysis using antibodies specific for CBP and p100 confirm the presence of both proteins in cell lysates from E 11.5 limb buds. (F) Streptavidin-mediated precipitation of biotinylated LncRNA-HIT and U1 RNA transcripts reveals co-precipitation of CBP and p100 from limb mesenchyme, confirming recruitment of both proteins by the lncRNA in the limb mesenchyme. (G) RIP analysis of p100 and CBP from the limb mesenchyme reveals co-precipitation of endogenous LncRNA-HIT transcript, confirming that CBP, p100, and LncRNA-HIT are present in a complex in the limb bud mesenchyme. Values represent the mean fold enrichment of LncRNA-HIT after precipitation with antibodies specific for CBP, p100 from three independent assays. Control precipitations were performed in parallel using murine IgG. Bars represent the standard deviation of the mean from the three independent assays.