Co-localization of endogenous Htt with BDNF mRNA in rat cortical neurons. (a) Monoclonal α-Htt antibody (MAB2166, Millipore) is highly specific and demonstrates the broad subcellular distribution of full length Htt. Western blot of mouse brain fractions prepared by sequential centrifugation of total brain homogenate (see Methods) and probed with MAB2166. Htt is present in nuclear (P1), membrane-associated (P2), large cytoplasmic particles (P3), and soluble pools (S3) of protein. This broad distribution of Htt protein is in agreement to previously published observations seen with a different antibody (rabbit antibody AP78 against N-terminal peptide, ). (b) Co-localization of endogenous Htt (green) with endogenous BDNF mRNA (red) in DIV 8 rat cortical neurons. Scale bar: 10 μm. (c) A schematic of the MS2-based mRNA detection system. BDNF-3'UTR positioned downstream of MS2 binding sites is detected by the fluorescently tagged NLS-MS2-Venus protein by microscopy. (d-f) Co-localization of endogenous Htt with BDNF 3'UTR mRNA in rat cortical neurons. BDNF 3'UTR linked to MS2 is detected by the co-expressing NLS-MS2-Venus, which is shown in green, and endogenous Htt in red (Cy3). Htt co-localizing with BDNF mRNA appears yellow in merged images. BDNF mRNA reporter construct with short 3'UTR (400 bases), long 3'UTR (2.6 kb) and full-length 3'UTR (3 kb) are shown in (d), (e) and (f), respectively. Scale bar: 10 μm, P: proximal.