Journal: Cell Death & Disease

Article Title: The Yersinia Type III secretion effector YopM Is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3

doi: 10.1038/cddis.2016.413

Figure Lengend Snippet: Y. pestis T3SS YopM is an E3 ubiquitin ligase. ( a and b ) Immunoblotting showing autoubiquitination by GST-YopM in the presence ( a ) or absence ( b ) of E1 and E2, ATP, and Ub. ( c ) Effects of mutations of Cys 68 on YopM–catalyzed polyubiquition chain synthesis. Ubiquitination assays were carried out using GST-YopM and GST-YopM C68A. All cell-based experiments were performed independently two to three times with comparable results

Article Snippet: In vitro ubiquitination assays (Enzo Ubiquitinylation Kit) were performed in 50 μ l reaction mixture containing reaction buffer (25 mM Tris-HCl at pH 7.5, 50 mM NaCl, 5 mM ATP, 10 mM MgCl 2 and 0.1 mM DTT), 2.5 μ l E1, 5 μ l mixed E2, and 2.5 μ l ubiquitin purified in the presence or absence of GST-YopM or GST-YopM C68A at the indicated times.

Techniques: Ubiquitin Proteomics, Western Blot

Journal: Cell Death & Disease

Article Title: The Yersinia Type III secretion effector YopM Is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3

doi: 10.1038/cddis.2016.413

Figure Lengend Snippet: YopM mediates K63-linked ubiquitination of NLRP3. ( a ) In vitro ubiquitylation assay with Flag-NLRP3 and a mixture of E1, E2s, ATP, ubiquitin, GST-YopM, or GST-YopM C68A. GST-YopM and Flag-NLRP3 were used to show equal loading. ( b ) Immunoblotting showing ubiquitination levels of NLRP3 in HEK293 cells transfected with Flag-NLRP3, Myc-YopM, and HA-tagged ubiquitin, HA-tagged K48 ubiquitin (Ub K48) or HA-tagged K63 ubiquitin (Ub K63), treated with MG132 (10 μ M) for 12 h. ( c ) Immunoblotting showing ubiquitination levels of endogenous NLRP3 in HEK293 cells transfected with HA-tagged ubiquitin together with different doses of Myc-YopM. ( d ) Immunoblotting showing ubiquitination levels of endogenous NLRP3 in BMDMs transfected with HA-tagged ubiquitin together with Myc-YopM or Myc-YopM C68A. ( e ) Immunoblotting showing ubiquitination level of NLRP3 in HEK293 cells transfected with Flag-NLRP3, HA-tagged K63 ubiquitin together with Myc-YopM, Myc-YopM C68A, or Myc-YopM ΔN. ( f ) BMDMs cells transfected with Flag-YopM or Flag-YopM C68A were infected Y. pestis ΔYopM (MOI=20). Cell extracts were prepared at the indicated time points and anti-NLRP3 immunoprecipitates were analyzed by immunoblotting with anti-Ub antibody. ( g ) BMDMs were infected with different background of Y. pestis . After infection, cell extracts were prepared at the indicated time points and anti-NLRP3 immunoprecipitates were analyzed by immunoblotting with anti-Ub antibody. Cell-based studies were performed independently three times with comparable results

Article Snippet: In vitro ubiquitination assays (Enzo Ubiquitinylation Kit) were performed in 50 μ l reaction mixture containing reaction buffer (25 mM Tris-HCl at pH 7.5, 50 mM NaCl, 5 mM ATP, 10 mM MgCl 2 and 0.1 mM DTT), 2.5 μ l E1, 5 μ l mixed E2, and 2.5 μ l ubiquitin purified in the presence or absence of GST-YopM or GST-YopM C68A at the indicated times.

Techniques: Ubiquitin Proteomics, In Vitro, Ubiquitin Assay, Western Blot, Transfection, Infection

Journal: Oncogenesis

Article Title: Stabilization of FASN by USP5-mediated deubiquitination promotes hepatocellular carcinoma progression

doi: 10.1038/s41389-025-00589-8

Figure Lengend Snippet: A Co-immunoprecipitation followed by mass spectrometry analysis of HEK-293T cells expressing exogenous USP5 identified 896 interacting proteins in total. The top five most abundant interacting proteins (excluding USP5 itself) are presented. B Comparative analysis revealed that among 650 proteins showing increased ubiquitination upon USP5 knockdown (by ubiquitin proteomics), 126 overlapped with the USP5-interacting proteins identified (Venn diagram representation). C Interaction between USP5 and FASN was confirmed by co-immunoprecipitation (co-IP) in both Huh7 and SK-Hep-1 cell lines. D Western blot analysis demonstrated that USP5 knockdown or overexpression significantly altered FASN protein levels.

Article Snippet: The ubiquitination proteomics was performed by Novogene (Beijing, China).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Ubiquitin Proteomics, Knockdown, Co-Immunoprecipitation Assay, Western Blot, Over Expression

Journal: Oncogenesis

Article Title: Stabilization of FASN by USP5-mediated deubiquitination promotes hepatocellular carcinoma progression

doi: 10.1038/s41389-025-00589-8

Figure Lengend Snippet: A The control and USP5 knockdown Huh7 and SK-Hep-1 cells were treated with vehicle or the proteasome inhibitor MG132 (10 μM) for 10 h, and whole-cell lysates were prepared and subjected to immunoblotting. FASN was detected with β-actin serving as the loading control. B Cycloheximide (CHX, 20 μg/mL) chase experiments in control and USP5-knockdown cells showed time-dependent changes in FASN protein stability (upper: representative blots of FASN and β-actin; bottom: quantification of FASN/β-actin ratios). C , D Huh7 ( C ) and SK-Hep-1 ( D ) cells with USP5 knockdown or overexpression were treated with MG132 (10 μM, 10 h), followed by immunoprecipitation with an anti-FASN antibody and immunoblotting with the indicated antibodies. The levels of FASN ubiquitination were quantified. E Representative IHC staining of USP5 and FASN in sequential tissue sections (left). Scatter plot showing coordinated expression patterns across specimens, with statistical significance determined by Pearson correlation analysis (right).

Article Snippet: The ubiquitination proteomics was performed by Novogene (Beijing, China).

Techniques: Control, Knockdown, Western Blot, Over Expression, Immunoprecipitation, Ubiquitin Proteomics, Immunohistochemistry, Expressing