Figure EV3. Poly‐GA inclusions sequester the proteasome.

• A, B
  Immunofluorescence of the proteasome subunit PSMC4 and poly‐GA inclusions in GA₁₇₅‐GFP‐transfected HeLa cells and GA₁₇₅‐GFP‐transduced rat primary neurons. To confirm cell viability, the cytoplasm of HeLa cells was stained with HCS CellMask™ Deep Red Stain and neuronal dendrites were labeled with MAP2. Scale bar denotes 20 μm.
• C, D
  Immunoblots of HeLa cells that were co‐transfected with HA‐ubiquitin and GFP, GA₁₇₅‐GFP, GFP‐GR₁₄₉, PR₁₇₅‐GFP, GFP‐GP_47, and PA₁₇₅‐GFP and incubated for 48 h and analyzed by densitometry. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. **P < 0.01.
• E–H
  Flow cytometry analysis of non‐cell‐autonomous proteasome inhibition using HEK293 Ub_G76V‐GFP reporter cells co‐cultured for 48 h with GA₁₇₅‐RFP‐ or RFP‐transfected cells. (E) Two‐color scatter plots, presented as pseudo‐color density plots, with compensated RFP fluorescence plotted on the x‐axis and compensated GFP fluorescence on the y‐axis. A representative experiment out of three independent repeats is shown. (F) Comparisons of the corresponding histograms for compensated RFP and Ub_G76V‐GFP fluorescence from one representative experiment that shows specific transmission of GA₁₇₅‐RFP associated with accumulation of Ub_G76V‐GFP in cells co‐cultured with GA₁₇₅‐RFP. (G) Accumulation of Ub_G76V‐GFP signal in non‐transfected receiver cells that were co‐incubated for 48 h with GA₁₇₅‐RFP‐transfected donor cells compared with RFP control. n = 3 biological replicates. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t‐test with Welch's correction. **P < 0.01. (H) Histograms for Ub_G76V‐GFP intensity showing separate analysis of RFP‐positive and RFP‐negative receiver cells for the GA₁₇₅‐RFP condition. RFP gating as indicated in (F).

Source data are available online for this figure.