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. 2026 Aug 6;23(5):e00970. doi: 10.1016/j.neurot.2026.e00970

Fig. 7.

Fig. 7

Isg15 and C9orf72 impair proteasomal degradation and promote accumulation of ubiquitinated proteins. (a, b) N2a cells were co-transfected with GFP-mHTT and Ubi-HA plasmids, with or without Isg15 overexpression, and analyzed 48 h post-transfection by immunoblotting. (a) Representative immunoblots showing HA-tagged ubiquitinated proteins, polyubiquitinated proteins, Isg15, and γ-tubulin. (b) Quantification of HA-tagged ubiquitinated proteins normalized to γ-tubulin, showing increased ubiquitinated protein accumulation upon Isg15 overexpression. (n = 4). (c–e) Analysis of proteasomal degradation using the UbiG76V-GFP reporter. N2a cells were co-transfected with GFP-mHTT and UbiG76V-GFP, with or without Isg15 overexpression. (c) Representative fluorescence images showing Hoechst-stained nuclei (blue) and UbiG76V-GFP (green). Increased reporter fluorescence indicates reduced proteasomal degradation in Isg15-overexpressing cells. Scale bar = 200 μm. (d) Representative immunoblots showing UbiG76V-GFP, ubiquitin, Isg15, and γ-tubulin. (e) Quantification of UbiG76V-GFP levels normalized to γ-tubulin, showing increased reporter accumulation upon Isg15 overexpression. (n = 4). (f, g) N2a cells were co-transfected with GFP-mHTT and Ubi-HA, with or without C9orf72 overexpression, and analyzed 48 h post-transfection. (f) Representative immunoblots showing HA-tagged ubiquitinated proteins, polyubiquitinated proteins, C9orf72, and γ-tubulin. (g) Quantification of HA-tagged ubiquitinated proteins normalized to γ-tubulin, showing increased ubiquitinated protein accumulation upon C9orf72 overexpression. (n = 5). (h–j) Analysis of proteasomal degradation using the UbiG76V-GFP reporter under C9orf72 overexpression conditions. (h) Representative fluorescence images showing Hoechst-stained nuclei (blue) and UbiG76V-GFP (green). Increased reporter fluorescence indicates reduced proteasomal degradation in C9orf72-overexpressing cells. Scale bar = 200 μm. (i) Representative immunoblots showing UbiG76V-GFP, ubiquitin, C9orf72 and γ-tubulin. (j) Quantification of UbiG76V-GFP levels normalized to γ-tubulin, showing increased reporter accumulation upon C9orf72 overexpression. (n = 5). Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by unpaired t-test.