Figure 3.

Application of TAMRA-Ub to study ubiquitination of mHtt aggregates (A) Scheme of TAMRA-Ub with Ub being fluorescently labeled on the N-terminus with TAMRA (5-tetramethylrhodamine, excitation 550 nm, emission 590 nm). (B) Electroporation of adherent cells with TAMRA-Ub dissolved in mannitol buffer, placed on ice to prevent intravesicular staining due to uptake by endocytosis. (C) TAMRA-Ub electroporated into cells is mainly localized in the nucleus and present on vesicles, while upon mHtt expression most TAMRA-Ub is recruited into the mHtt aggregate. (D) Upon electroporation of TAMRA-Ub, cells can be lysed and aggregates can be solubilized and separated by SDS-PAGE, with fluorescent bands representing TAMRA-Ub conjugated proteins.