Figure 3

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 μM) 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