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.
