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