Toxin B efficiently glucosylates Cdc42 and Rac without affecting LPS-mediated SEK and JNK activation.

(A) The Rho-family GTPase inhibitor toxin B does not affect JNK activation by LPS; quiescent BAC-1.2F5 cells were incubated with toxin B (Tox B, 100 ng/mL) for 60 minutes before stimulation with 1.5 μg/mL LPS for 15 minutes. The presence of phosphorylated forms of SEK and JNK, and of JNK1 as a loading control, was detected by immunoblotting with the corresponding antibodies. (B) In vivo treatment with toxin B efficiently glucosylates Rho subtype proteins in BAC-1.2F5 cells. Cells were either left untreated or pretreated with toxin B (100 ng/mL for 60 minutes). Lysates (100 μg) or recombinant GST-Rac (1 μg) were incubated in vitro with recombinant toxin B (fragment CDB546) and UDP-[¹⁴C]glucose (20 μmol/L) for 60 minutes at 37°C. Glucosylated proteins were analyzed by SDS-PAGE and phosphorimaging. The identity of the GTPases was confirmed by immunoblotting.