Journal: bioRxiv

Article Title: Novel VCP activator reverses multisystem proteinopathy nuclear proteostasis defects and enhances TDP-43 aggregate clearance

doi: 10.1101/2023.03.15.532082

Figure Lengend Snippet: (A, B) Immunofluorescence confocal images of HeLa cells expressing TDP-4FL treated with 4μM MG132 for 0, 3, or 6 hours stained for myc (TDP-4FL, red) and either ubiquitin (A, green) or VCP (B, green). Scale bar, 10µm. (C) Immunoblots for myc (TDP-4FL) from soluble and insoluble protein fractions from HeLa cells expressing TDP-4FL treated with 4μM MG132 for 0, 3, or 6 hours. GAPDH provided as a loading control. (D-E) Quantification of (D) soluble or (E) insoluble TDP-4FL immunoblots. (n = 4 independent experiment, results are expressed as mean ± SEM over time. One-way ANOVA, ** P < 0.01 for soluble protein, *P < 0.05 for insoluble protein). (F) Anti-myc immunoprecipitation from insoluble protein fractions, immunoblotted for myc (TDP-4FL, red) and ubiquitin (green).

Article Snippet: Primary antibodies used include mouse anti-Myc (9E10), rabbit anti-Myc (ab9106, Abcam, Boston, MA), rabbit anti-TDP-43 (C2089, gift from CNDR), mouse anti-VCP (NB120-11433, Novus Biologicals, Centennial, CO, USA), rabbit anti-ubiquitin (43124, Cell Signaling Technology, Danvers, MA), and rabbit anti-GAPDH (2118, Cell Signaling Technology).

Techniques: Immunofluorescence, Expressing, Staining, Western Blot, Immunoprecipitation