proteasome inhibitor mg132  (MedChemExpress)

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: Spastin is ubiquitinated. (A) Ubiquitin peptides (red font) detected by MS analysis of spastin precipitates from rat brain lysate. (B) GST-ubiquitin was purified and used for GST pulldown of proteins from rat brain lysate. (C) Ubiquitin was immunoprecipitated from rat brain lysate and immunoblotted with an anti-spastin antibody. (D) Spastin was immunoprecipitated with an anti-spastin antibody and immunoblotted with an anti-ubiquitin antibody. (E) Flag-Ub and GFP-spastin were overexpressed in HEK293 cells. The cells were treated with the proteasome inhibitor MG132 (20 µM) for 8 hours, then collected and lysed. GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (F) GFP-Spastin was overexpressed in COS7 cells and HEK293 cells. The cells were treated with CHX (10 µg/mL) for 24 hours and MG132 for 8 hours, and spastin expression was detected. (G) COS7 cells and HEK293 cells were treated with CHX and MG132, and endogenous spastin was detected. (H) HT22 cells were treated with CHX and MG132 and collected at the indicated times for western blotting. Quantification of spastin levels relative to GAPDH is shown. (I) Flag-Ub and Flag-tagged ubiquitin mutants (K48R/K63R) were overexpressed with GFP-spastin in HEK293 cells. Spastin was detected by western blotting. (J) Flag-Ub and Flag-tagged ubiquitin mutants (K48R/K63R) were overexpressed with GFP-spastin in HEK293 cells. Cells were treated with MG132 for 8 hours before collection. Then, GFP-spastin was immunoprecipitated with anti-GFP antibody and immunoblotted with an anti-Flag antibody. n = 3. Data are shown as mean ± SEM. * P < 0.05. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; GST: glutathione-S-transferase; IP: immunoprecipitation; MS: mass spectrum.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Ubiquitin Proteomics, Purification, Immunoprecipitation, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: Spastin ubiquitination sites. (A) Overview of wild-type and truncated spastin. (B) The truncated spastin mutants were expressed in HEK293 cells that were treated with CHX and MG132, followed by western blotting for spastin. (C) The truncated spastin mutants were co-expressed with Flag-Ub in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. Then, GFP was immunoprecipitated with an anti-GFP antibody and immunoblotted with anti-Flag antibody. (D) Potential spastin ubiquitination sites (K219/K386/K484/K497/K519) were predicted using the PhosphoSite tool ( https://www.phosphosite.org/ ). (E) WT or 5KR was expressed in HEK293 cells, which were collected at the indicated times for western blotting. (F) Quantification of spastin levels relative to GAPDH. (G) GFP-labeled wild-type spastin and spastin 5KR were co-expressed with Flag-Ub in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. Then, GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (H) The wild-type and mutant spastin plasmids were transfected into HEK293 cells, which were then treated with CHX. Spastin was detected by western blot at the indicated times. Quantification of spastin levels relative to GAPDH is shown. n = 3. Data are shown as mean ± SEM. * P < 0.05. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; IB: immunoblotting; IP: immunoprecipitation; spastin 5KR: spastin with all five sites mutated; WT: wild-type.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Phospho-proteomics, Labeling, Mutagenesis, Transfection

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: Cullin3 mediates spastin ubiquitination. (A) Cullin (Cullin3/Cullin4b/Cullin5) peptides were detected by MS analysis of spastin precipitates from rat brain lysates. (B) Flag-tagged Cullins (Cullin3/Cullin4b/Cullin5) were co-expressed with GFP-spastin in HEK293 cells. GFP-spastin expression was detected for western blotting. (C) Increasing amounts of Flag-Cullin3 were co-expressed with GFP-spastin in HEK293 cells, and GFP-spastin expression was detected. (D) Flag-Cullin3 was co-expressed with GFP-spastin in HEK293 cells for 24 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (E) HA-Cullin3 was co-expressed with GFP-spastin in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody, and ubiquitinated spastin was detected by immunoblotting using an anti-Flag antibody. (F) GFP-spastin and Flag-Cullin3 were co-expressed in HEK293 cells that were then treated with CHX. The cells were collected at the indicated times for western blotting. The lysates were blotted with anti-GFP and anti-Flag antibodies. n = 3. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; HA: hemagglutinin; IP: immunoprecipitation.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Neural Regeneration Research

Article Title: The Cullin3–Ring E3 ubiquitin ligase complex and USP14 regulate spastin-mediated microtubule severing and promotion of neurite outgrowth

doi: 10.4103/NRR.NRR-D-25-00037

Figure Lengend Snippet: USP14 deubiquitinates and stabilizes spastin. (A) USP14 peptides were detected by MS analysis of spastin precipitates from rat brain lysate. (B) Increasing amounts of GFP-USP14 were co-expressed with Flag-spastin in HEK293 cells, and Flag-spastin expression was detected. (C, D) GFP-USP14 and Flag-spastin were co-expressed in HEK293 cells that were then treated with CHX. The cells were collected at the indicated times for western blotting. The lysates were collected and blotted with anti-Flag and anti-GFP antibodies. (E) Flag-USP14 was co-expressed with GFP-spastin in HEK293 cells for 24 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (F) Flag-spastin was co-expressed with GFP-USP14 in HEK293 cells for 24 hours before collection. GFP-USP14 was immunoprecipitated with an anti-GFP antibody and immunoblotted with an anti-Flag antibody. (G) HA-USP14 was co-expressed with GFP-spastin in HEK293 cells. The cells were treated with MG132 for 8 hours before collection. GFP-spastin was immunoprecipitated with an anti-GFP antibody, and ubiquitinated spastin was detected with an anti-Flag antibody. n = 3. CHX: Cycloheximide (a translational inhibitor that blocks protein synthesis); GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; IB: immunoblotting.

Article Snippet: 293T cells were co-transfected with the specified plasmids and a plasmid encoding FLAG-tagged ubiquitin for 48 hours, then treated with the proteasome inhibitor MG132 (20 μM, MedChemExpress, Cat# HY-13259, Monmouth Junction, NJ, USA) for 12 hours to inhibit proteasome activity and prevent degradation of ubiquitinated proteins.

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: Translational Vision Science & Technology

Article Title: Pathogenicity and Functional Analysis of Multi-Variant Allele of RPE 65 Causing Retinitis Pigmentosa

doi: 10.1167/tvst.15.2.1

Figure Lengend Snippet: Pathogenic variant damaged the stability of RPE65 possibly through the ubiquitination–proteasome pathway. ( A ) When HEK293T cells were transfected with WT variant plasmids for 24 hours and treated with 100-µM CHX, RPE65 expression was detected. ( B ) RPE65 stability was detected after cells were treated with 30-µM MG-132. ( C ) Twenty-four hours after transfection, WT mutant proteins were enriched by immunoprecipitation, and their ubiquitination levels were detected.

Article Snippet: After 24 hours of transfection, HEK293T cells were treated with 100-μM cycloheximide (CHX, HY-N0901; MedChemExpress, Monmouth Junction, NJ) or CHX mixed with MG-132 proteasome inhibitor (HY-12320; MedChemExpress).

Techniques: Variant Assay, Ubiquitin Proteomics, Transfection, Expressing, Mutagenesis, Immunoprecipitation