Fig. 4.

Unc119 specifically co-aggregates with poly-GA. a Quantitative proteomics of GFP immunoprecipitations from primary cortical neurons transduced with GFP or GA₁₄₉-GFP (DIV6 + 17). p62/Sqstm1 shows highest enrichment and statistical significance. Unc119 was identified by two unique peptides (GGGGTGPGAEPVPGASNR and LGPLQGK) and one peptide (YQFTPAFLR) shared with its homolog Unc119b. Full protein names are listed in Table 1. Upper panel illustrates distribution of quantified protein abundances binned for enrichment factors (x-axis below). Enrichment of ubiquitin-related and proteasomal proteins in the poly-GA interactome is highlighted in green. Lower panel depicts volcano plot showing poly-GA interacting proteins. False discovery rate (FDR) controlled statistical analysis identified 20 poly-GA interacting proteins compared to control (red dots). Dotted line depicts threshold for statistical significance. b Immunofluorescence of HEK293 cells co-transfected with GFP or GA₁₇₅-GFP and HA-Unc119 or empty vector control (ctrl). Staining with HA and GFP antibodies and DAPI as nuclear marker. Many GA₁₇₅-GFP inclusions show co-aggregation of HA-Unc119 (examples marked with arrows). Separate channels of these images are shown in Fig. S6. Scale bar 30 µm. c HEK293 cells were transfected with the five different poly-DPR constructs (GA₁₇₅-GFP, GFP-GR₁₄₉, PR₁₇₅-GFP, PA₁₇₅-myc and GP₈₀-V5) or Q₁₀₂-GFP and analyzed using GFP fluorescence and immunostaining of HA-Unc119, PA₁₇₅-myc and GP₈₀-V5 using specific antibodies 2 days later. HA-Unc119 co-aggregates only with GA₁₇₅-GFP (white arrows). DAPI (in blue) was used as a nuclear marker. Scale bar 20 µm