Extended Data Figure 9: Pyrin requires, but does not directly interact with, the Rho subfamily in mediating TcdB-induced inflammasome activation.
From: Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome
a–d, Effects of siRNA knockdown of RHOA, B and C on TcdB-induced ASC foci formation. 293T cells stably expressing Pyrin and RFP–ASC were transfected with indicated siRNA or siRNA combinations followed by TcdB stimulation. Rhoa-1/2, Rhob-1/2 and Rhoc-1/2 are two independent siRNAs targeting RHOA, B and C, respectively. qRT–PCR analyses of the knockdown efficiency are shown in a, c (n = 3; mean ± s.d.) and percentages of cells showing ASC foci formation are in b, d (n = 3, mean ± s.d.; P value, Student’s t-test). e, f, Co-immunoprecipitation interaction assays of Pyrin and Rho GTPases. 293T cells were transfected with haemagglutinin (HA)–6myc–PyrinΔN88 (deletion of N-terminal 88 residue.) together with Flag–6myc–PyrinΔN88 or an indicated Rho GTPase construct in e. DC2.4 cells stably expressing Flag–6myc–PyrinΔN88 were left untreated or stimulated with TcdB in f. Immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (input) shown in e and f are representative from at least three repetitions.
