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. Author manuscript; available in PMC: 2010 Apr 10.
Published in final edited form as: Mol Cell. 2009 Apr 10;34(1):93. doi: 10.1016/j.molcel.2009.03.008

Figure 6. Importance of the Fic domain in H. somni pathogenesis.

Figure 6

(A) CCS from H. somni 2336 but not H. somni 129Pt is immunoreactive against anti-Fic2. CCS from 2336 and 129Pt were separated on SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antibody raised against Fic2. Only CCS from 2336 was immunoreactive with anti-Fic2.

(B) CCS from 2336 adenylylates Rho GTPases. CCS from 2336 and 129Pt were used in an in vitro adenylylation reaction containing GST-RhoA, GST-Rac or GST-Cdc42. Samples were separated by SDS-PAGE and visualized by autoradiography. 2336 CCS adenylylates purified Rho GTPases.

(C) CCS from 2336 adenylylates the endogenous Rho GTPases present in HEK293T cell extract. CCS from 2336 or 129Pt was incubated with HEK293T cell extract in an in vitro adenylylation reaction. Samples were separated by SDS-PAGE, Coomassie stained and visualized by autoradiography. The migration of endogenous GTPases was determined by Western analysis using antibody to Cdc42. 2336 CCS adenylylates Rho GTPases in cellular extract.

(D) Inhibition of HeLa cell cytotoxicity with antiserum to Fic2 or convalescent serum from H. somni infected animals. Treatment of HeLa cells with CCS or live H. somni (black bars) induces cytotoxicity as compared to DMEM treated cells (white bars). Pretreatment of H. somni or CCS with preimmune rabbit serum did not reduce toxicity (grey bars). Treatment with rabbit anti-Fic2 serum (red bars) or convalescent bovine serum (blue bars) caused a significant decrease in the cytotoxic HeLa cells. Asterisk (*) denotes significant (p value ≤ 0.05) mean difference from cells without serum or those treated with preimmune serum. All groups were significantly different from untreated (medium) control group.