Figure 1.
Pull-down assays with GST-YopH D356A.
A, HEK293 cells expressing different proteins, either untreated (control) or treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed, were lysed and probed for interaction with GST-YopH D346A or GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with GST-YopH D356A was detected by Western blot with specific antibodies for Lck or Vav, and with anti-HA antibody for other proteins. B, As in A, HEK293 cells, expressing the same proteins and treated with PV, were lysed and probed for interaction with two different amounts of GST-YopH D346A (5 and 2 µg) or GST (5 µg each) as a negative control in pull-down assays. GST and GST-YopH D346A fusion protein used in these assays are shown at the lower panel from one representative blot. TL denotes total lysates of the transfected cells and corresponds to a 10% of the amount used for each pull-down assay. Assays were done independently for each protein.
Figure 2.
YopH dephosphorylation assay of several proteins.
A, Dephosphorylation assay for HA-Gab1, HA-Gab2 and Vav at different time-points, using 1 µg of GST-YopH or GST-YopH D356A. The assay was stopped by addition of sample buffer, and after SDS-PAGE, samples were transferred to nitrocellulose and tyrosine phosphorylation was detected by Western blot with anti-phosphotyrosine antibody. B, Dephosphorylation assay for HA-Fyb, Lck and HA-p85 was carried out as in A, but using shorter incubation times. Proteins used as substrates in these assays were obtained from HEK293 transfected with the corresponding plasmids and treated with pervanadate. Proteins were immunoprecipitated and distributed equally in different tubes for the several time-points of the assay.
Figure 3.
Pull-down assays with different deletion mutants of YopH.
A, Schematic diagram showing the different deletion mutants of YopH used in this study. B, HEK293 cells expressing different proteins and treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed were lysed and probed for interaction with GST-YopH D/A (mutation D346A), the different deletion mutants shown in A fused to GST, and GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with YopH fragments was detected by Western blot with specific antibodies for Lck and Vav, and with anti-HA antibody for other proteins. An independent experiment was done for each protein. The lower panel shows a representative blot from one of the experiments to show that similar amounts of GST proteins were used in these assays.
Figure 4.
Inhibition of different luciferase transcriptional reporters by YopH deletion mutants.
A, Activation of a luciferase reporter gene driven by NFAT/AP1. B, Activation of a NF-κB-driven luciferase reporter gene. C, Activation of a luciferase reporter gene driven by IL-2 promoter. In all cases, the data represent the mean±S.D. from triplicate determinations. *, p<0.05, **, p<0.005, ***, p<0.0005 as compared with pEF5 trasfected cells stimulated through TCR and CD28.