FIGURE 5.

Detection of the cp-2 fragment by a specific antibody to the neo-epitope Arg¹⁶⁷ of Htt in HEK293 cells. A, Western blot of total cell extracts (30 μg/lane) from HEK293 cells transfected with the indicated Htt constructs, or an empty vector (Mock). Truncated FLAG-Htt fusion proteins and cp-1/cp-2 fragments (marked by arrows) were detected with antibodies to FLAG (left panel). The Htt-N167-82Q fragment migrates on a gel more slowly than the cp-2 fragment derived from Htt-N511-52Q, consistent with the length of poly(Q). Antibody to Arg¹⁶⁷ neo-epitope detects the Htt-N167 fragment and the cp-2 fragment, has low cross-reactivity with the Htt-N171 fragment, and fails to detect Htt-N511 fragment or any fragments of Htt-Δ167-170 (right panel, data shown for NE167 antibody). B, Western blot of total cell extracts from HEK293 cells transfected with the indicated Htt constructs or an empty vector (Mock). Htt proteins (indicated by arrows) were detected with an antibody to exon 1 of Htt (left panel, 20 μg per lane). The cp-2 fragments produced from Htt-N511 or Htt-N586 were specifically detected by an antibody to the Arg¹⁶⁷ neo-epitope (right panel, 40 μg/lane). Blots, shown in A and B, were re-probed with an antibody to actin for loading control. C, Western blot of formic acid-soluble aggregate fractions from HEK293 cells transfected with indicated constructs or an empty vector (Mock). Htt fragments were detected with antibodies to exon 1 (left panel) or to the Arg¹⁶⁷ neo-epitope (right panel) of Htt.