lfhp 1c  (MedChemExpress)

Journal: International Journal of Biological Sciences

Article Title: METTL3-mediated m6A modification of OTUD1 aggravates press overload induced myocardial hypertrophy by deubiquitinating PGAM5

doi: 10.7150/ijbs.95707

Figure Lengend Snippet: OTUD1 directly binds to PGAM5. (A) Schematic illustration of mass spectrometry analysis to identify proteins binding to OTUD1. (B) PGAM5 peptides derived from the mass spectrometric analysis of OTUD1. (C-D) Co-IP assays of the interaction between OTUD1 and PGAM5 in 293T cells transfected with the indicated plasmids. (E-F) Co-IP assays of Otud1 and Pgam5 in NRCMs. (G-H) Co-IP assays of Otud1 and Pgam5 in heart tissues. (I) Schematic illustration of the OTUD1 domain deletion mutants. (J) Co-IP analysis of the binding region of OTUD1 and PGAM5.

Article Snippet: To test the role of PGAM5 in pathological cardiac hypertrophy, we used PGAM5 inhibitor LFHP-1c (HY-139598, MCE) in TAC mice.

Techniques: Mass Spectrometry, Binding Assay, Derivative Assay, Co-Immunoprecipitation Assay, Transfection

Journal: International Journal of Biological Sciences

Article Title: METTL3-mediated m6A modification of OTUD1 aggravates press overload induced myocardial hypertrophy by deubiquitinating PGAM5

doi: 10.7150/ijbs.95707

Figure Lengend Snippet: OTUD1 deubiquitinates PGAM5. (A) Representative western blot of Pgam5 protein in heart of Flox and Otud1-CKO mice subjected to Sham or TAC (n=3). (B) Representative western blotting result of Pgam5 in NRCMs infected with Ad-GFP and Ad-Otud1 and treated with Ang II (10 μM) for 48h and cycloheximide (CHX, 50 μM) for the indicated time points (n=3). (C) Results of ubiquitination assays confirming the ubiquitination of PGAM5 after overexpression of OTUD1 for 48 h and treated with MG132 (50 μM) for 6 h in 293T. (D) Results of ubiquitination assays confirming the K48 ubiquitination of PGAM5 after co-transfected with Flag-OTUD1, HA-Ub-WT, HA-Ub-K48, HA-Ub-K63 for 24 h and treatment with MG132 (50 μM) for 6 h in 293T. (E) Results of ubiquitination assays confirming the expression and ubiquitination of Pgam5 in NRCMs infected with Ad-GFP and Ad-Otud1 and followed by Ang Ⅱ treatment. (F) Results of ubiquitination assays confirming the expression and ubiquitination of Pgam5 in heart tissue from Flox and CKO mice subjected to sham or TAC. (G) Results of ubiquitination assays confirming the ubiquitination of Pham5 after overexpression of OTUD1, OTUD1(C320A) and Pgam5 for 24 h and treated with MG132 (50 μM) for 6 h in 293T cells. (H) Immunoblotting analysis of Pgam5, Ad-Otud1 and Ad-Otud1 (C294S) in NRCMs.

Article Snippet: To test the role of PGAM5 in pathological cardiac hypertrophy, we used PGAM5 inhibitor LFHP-1c (HY-139598, MCE) in TAC mice.

Techniques: Western Blot, Infection, Over Expression, Transfection, Expressing