Immunofluorescent analysis of formalin fixed, permeabilized 293T cells transfected with Huntingtin23Q (left panel) and Huntingtin148Q (right panel)stop constructs ending in amino acid 586 labeling capase cleaved Huntingtin with ab155930 at 1/50(green). Nuclei were stained with Hoechst 33342 (blue).
All lanes : Anti-Huntingtin antibody (ab155930) at 1/500 dilutionLane 1 : Lysate containing endogenous Huntingtin without capase activityLane 2 : Lysate containing endogenous Huntingtin with capase2 activityLane 3 : Lysate containing endogenous Huntingtin with capase3 activityLane 4 : Lysate containing endogenous Huntingtin with capase6 activityLane 5 : Lysate containing endogenous Huntingtin with capase7 activityLane 6 : Lysate containing overexpressed recombinant Huntingtin fragment (aa1-513)Lane 7 : Lysate containing overexpressed recombinant Huntingtin fragment (aa1-536)Lane 8 : Lysate containing overexpressed recombinant Huntingtin fragment (aa1-552)Lane 9 : Lysate containing overexpressed recombinant Huntingtin fragment (aa1-586)Lysates/proteins at 20 µg per lane.SecondaryLanes 1, 2, 3, 4, 5, 6, 7 & 9 : Goat anti-rabbit HRP conjugated antibody at 1/30000 dilutionLane 8 : Goat anti-rabbit HRP conjugated antibody at 1/30000 dilutiondeveloped using the ECL technique
Sandwich ELISA was performed with a monoclonal antibody to Huntingtin and ab155930 to determine the antigen concentration of the Huntingtin cleavage products. The curve represents a dose response for neo586 in 293T cells overexpressing the Huntingtin construct.