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. 2022 Dec 29;32(9):1511–1523. doi: 10.1093/hmg/ddac306

Figure 4.

Figure 4

Effects of p.G64R-DOK7 on protein solubility. (A and C) Representative western blotting and quantitative analysis of DOK7 and GAPDH without ß-actin (A) or with ß-actin (C) in detergent-soluble fractions (S) and total protein lysates (T), in transfected COS7 cells. MG132 (20 μM) was added 3 h before harvesting cells (A). Expression levels were normalized to that of GAPDH, and also to the ratio of wild-type (WT)-DOK7. Mean and SD (n = 3 experiments) are indicated with individual values in red dots. Multiple Student t-test was applied (ns, no significance, *P < 0.05, ****P < 0.0001). Triplicated (B) and representative (D and E) western blotting of DOK7, ß-actin and GAPDH in detergent–soluble (S) and insoluble (In) fractions, in transfected COS7 cells. (D) MG132 at 0–20 μM was added 3 h before harvesting cells. (E) MG132 at 20 μM was added at indicated time points. (F) Representative images of WT-DOK7 and p.G64R-DOK7 treated with or without 20 μM MG132 treatment for 3 h in transfected COS7 cells. Green and blue signals represent DOK7 and DAPI, respectively. Scale bar = 10 μm. (G) The ratio of transfected COS7 cells with aggregates in each experiment is indicated in red dot. Each experiment is an average of five visual fields and is comprised of at least 100 cells. Mean and SD (n = 3 experiments) are indicated. One-way ANOVA with Dunnett’s multiple comparison test (ns, no significance; ****P < 0.0001).