Bifunctional Anti-Huntingtin Proteasome-Directed Intrabodies Mediate Efficient Degradation of Mutant Huntingtin Exon 1 Protein Fragments
Figure 1
scFv-C4-PEST enhances degradation of GFP-tagged httex1 soluble and insoluble protein fragments.
ST14A cells were co-transfected with GFP-tagged httex1-25Q (httex1-25Q-GFP) or (httex1-72Q-GFP), plus either empty vector, scFv-C4, or scFv-C4-PEST plasmids. A. Representative live cell imaging depicting reduction of httex1-72Q-GFP and httex1-25Q-GFP fluorescence in the scFv-C4-PEST co-transfected groups. Phase contrast confirms uniform cell integrity. 48 h; bar = 20 µm. B. SDS-PAGE Western blot and densitometry probed for htt (EM48), quantified vs. actin. Monomeric soluble mhttex1 protein fragments were quantitatively reduced in scFv-C4-PEST vs. scFv-C4 co-transfected cells. (Mean ± SEM; *p<0.05 comparing httex1-25Q-GFP co-transfections; *p<0.05 comparing httex1-72Q-GFP co-transfections) Note that B shows only soluble httex1-72Q fragment levels, which are low unless intrabody is present. C. Agarose Gel Electrophoresis for Resolving Aggregates (AGERA) shows the decrease of detergent-insoluble material in httex1-72Q-GFP to scFv-C4-PEST co-transfected cells compared to other groups.