Figure 2

Primary neurons in culture and in brain produce and accumulate cpA from over-expressed htt1-400. (a, b) Western blot shows lysates from striatum of 3 mice injected with AAV-htt1-400-18Q and 4 mice injected with AAV-htt1-400-100Q probed with anti-huntingtin antibody Ab1. Arrowheads show the expressed, intact htt1-400-18Q (a) and htt1-400-100Q (b), and arrows indicate cpA and cpB. Short unlabeled arrow indicates an intermediate band (b). For (a) 4-12% Bis-Tris gel and full-length huntingtin serves as a loading control, and for (b) 4-20% Tris-glycine gel and blot was reprobed with tubulin as a loading control. (c) Lysates from infected mouse brain striatum were also analyzed on the same gel as lysates from HeLa cells expressing htt400-100Q ± the proteasome inhibitor epoxomicin (EPX) to identify cpA and cpB from mouse brain. SDS-PAGE run on 10% Tris-glycine gel. (d) Western blots of lysates from primary rat striatal neurons treated with lentivirus delivering cDNAs encoding htt1-400-18Q or htt1-400-100Q. Concentration of lentivirus in multiplicity of infection (moi) indicated at top. Arrowhead, intact htt1-400; arrows, cpA and cpB, open arrowhead, non-specific band. 4-12% Bis-Tris gels. (e) Western blots of lysates from primary rat cortical neurons treated with lentivirus (80 moi) delivering cDNAs encoding htt1-400-100Q. Arrowhead, intact htt1-400; arrows, cpA and cpB; open arrowhead, non-specific band. 3-8% Tris-acetate gel. Cells were treated with 5 M epoxomicin (EPX) or carrier (DMSO) for final 4 hours. (f) Western blots of lysates from primary cortical mouse neurons infected with lentivirus-PGK-htt1-171 (18Q or 100Q) then harvested post-infection at days indicated. Lysates from uninfected control "C" neurons are shown for comparison. Arrowheads, intact expressed protein; arrows indicate cpA. For 171-100Q, an intermediate band is also visible. 4-12% Bis-Tris gel for 18Q and 3-8% Tris-acetate gel for 100Q. Samples were run on the same gel, white line indicates removal of lanes.