Extended Data Figure 5: Toxins-induced modification of Rho GTPases and Rho inactivation by TcdB, TcsL, C3 and B. cenocepacia infection.

a, Lysates of TcdB or TcsL-treated Caspase 1^−/− BMDM cells were subjected to in vitro ADP-ribosylation reaction by purified C3 toxin. Anti-RHOA immunoblotting shows TcdB modification of RHOA, as suggested by its resistance to further modification by the C3 toxin. b, c, e, DC2.4 cells were stimulated with TcdB, TcsL or LFn-tagged C3 toxin in b, c, or infected with B. cenocepacia (WT or the Δhcp mutant) in e. Cell lysates were subjected to glutathione S-transferase (GST)–RBD (the Rho binding domain of human Rhotekin protein) (b, e) and GST-PBD (the Rac/Cdc42 (p21) binding domain of human p21 activated kinase 1 protein (c, e) pulldown assays to measure GTP-bound RHOA and Rac/Cdc42, respectively. d, Caspase 1^−/− BMDMs were delivered with V. parahaemolyticus VopS and the two FIC domains in H. somni IbpA (IbpA-Fic1/2) by the LFn-PA system. H/A, mutation of the FIC-domain catalytic histidine. Anti-RHOA immunoblotting shows the SDS–PAGE mobility shift of RHOA as a result of effector-catalysed adenylylation. Data in all panels are representative of at least three repetitions.