Fig 1: Effects of exotoxin GAP domain self-ADP-ribosylation and cross-ADP-ribosylation on RhoA GTPase activity. (A) First, in vitro ADP-ribosylation assays were performed using each exotoxin, with or without NAD+ supplementation, for 2 h. Following this, 100 ng of total exotoxin from the reactions was incubated with 100 ng of RhoA for 1 h, and GTP consumption was measured using a GTP-Glo Assay Kit. P values were calculated using Welch’s unpaired t-test, and exact values are reported. (B) The experiment was performed as in panel A, but using catalytically inactivated exotoxins as indicated. (C) The experiment was performed as in panel A, using only 25 ng of in vitro reaction exotoxins for incubation with 100 ng of RhoA.