Fig. 2.

VEEV-nsP3 associates with DDX1 and DDX3. A) U87MG cells were uninfected (Mock) or infected with rTC-83-nsP3-HA (MOI:10) for 1 h. Fresh media were added to the cells and at 6 hpi cell lysates were collected and quantified. Two milligrams of total protein from each sample was immunoprecipitated with an HA antibody or an isotype IgG control antibody. The immunoprecipitates were processed for LC/MS-MS. A total of 3 independent experiments were performed and each MS run was performed in duplicate to generate a data set that was compared and contrasted for unique proteins identified only in the rTC-83-nsP3-HA infected samples immunoprecipitated with HA. The identified proteins were tiered based on at least 50% reproducibility and additionally tiered according to peptide hits. B) U87MG cells were infected with rTC-83-nsP3-HA (MOI:20) for 1 h. Fresh media were added to the cells and at 6 hpi cell lysates were collected and quantified. Two milligrams of total protein was immunoprecipitated with an isotype IgG control or an HA antibody. The immunoprecipitates were resolved by SDS-PAGE and the subsequent immunoblot probed with antibodies to DDX1, DDX3 or HA. The western blot is a representative image of 2 independent experiments.