Journal: bioRxiv

Article Title: USP14 regulates pS129 α-synuclein levels and oxidative stress in human SH-SY5Y dopaminergic cells

doi: 10.1101/2024.05.09.592905

Figure Lengend Snippet: Control SH-SY5Y and USP14-deleted cells were cultured for 48h as described in Methods ( A, B, C ). They were then treated for 5h with 20µM MG132, or DMSO as negative control, and analyzed further as indicated below. (A) Native-gel electrophoresis followed by in-gel activity assay was done as described in Methods for 26S/30S proteasome and 20S CP chymotrypsin-like activity. Left panels, UV-exposed native gel in the presence or absence of SDS to detect the activity of free 20S CP. Right panels, quantification of the densitometry ratio of 26S/30S proteasome, 20S CP activity without (W/O) or with SDS in USP14-deleted cells normalized to controls. Values are means ± S.E.M. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, n=3. (B) Native-gel electrophoresis followed by immunoblotting using a 20S antibody cocktail. Right panels, quantification of the densitometry ratio of 26S/30S and 20S complex in USP14-deleted cells normalized to controls. Values are means ± S.E.M. *p ≤ 0.05. n=4. (C) Immunoblotting using the antibody for 20S CP subunit proteins. Right panel, quantification of five 20S CP protein subunits normalized to β-actin. (D) Immunoblotting for K48-linked polyubiquitin chains antibody. Right panel, quantification of the K48-polyubiquitin smear normalized to β-actin. (C-D) Values are means ± S.E.M. **p ≤ 0.01, n=3.

Article Snippet: Standard protocol for wet-transfer was performed for 16h, 20V at 4°C followed by incubation with 20S (BML-PW8195, Enzo Lifesciences, USA), anti-USP14 antibody (J6111-6D6, Sigma, Germany).

Techniques: Cell Culture, Negative Control, Nucleic Acid Electrophoresis, Activity Assay, Western Blot