Figure 2. Low solubility proteasome substrates aggregate in a ubiquitin K63-dependent manner.

(A) Schematic diagram of the cell fractionation based on protein solubility (left). Wild-type SH-SY5Y cells were incubated with 20 µM MG132 or DMSO for 12 h prior to lysis with 0.5% NP40 buffer. Total cell lysates (T), detergent-soluble (S1) and insoluble (P1) fractions, and high-speed soluble (S2) and insoluble (P2) fractions were subjected to 4–20% SDS-PAGE and immunoblotting with an anti-ubiquitin antibody. (B) GFP-ubiquitin SH-SY5Y cells were treated with 20 µM MG132 for 12 h and analyzed as in A. (C) GFP-ubiquitin SHY5Y cells treated with 10 µM MG132 for 12 hours were imaged live by fluorescence confocal microscopy before and after photobleaching of on area in the cytoplasm (top) or in the inclusion (bottom). Typical images of SH-Y5Y GFP-ubiquitin cells before, immediately after and 30 and 60 seconds after photobleaching are shown. Color-coded pixel fluorescence intensities are shown in the right panel. Fluorescence intensities relative to the pre-treated area were reported for each time point (right). Data represent the mean ± SEM (n = 7 cytoplasm; n = 4 inclusions) of the normalized bleached region. (D) SH-SY5Y cells transiently transfected with the indicated GFP-ubiquitin constructs for 24 h prior to treatment with 5 µM MG132 for 8 h and PFA fixation with Hoechst. Two insets showing cells transfected with K63R-ubiquitin are shown to the right. (E) GFP-ubiquitin SH-SY5Y cells transiently transfected with the indicated HA-ubiquitin constructs for 24 h prior to treatment with 5 µM MG132 for 12 h and methanol fixation. Immuno-fluorescence was performed using mouse anti-HA and anti-mouse Alexa568 (Invitrogen) antibodies. All scale bars represent 10 µm.