FIG. 11.
Autoradiographic images of MBP-UL34 subjected to in vitro kinase assay with Us3 immunoprecipitates. (A) Vero cells were infected with wild-type YK304 (lane 1), YK515 (Us3S147A) (lane 2), or YK511 (Us3K220M) (lane 3) at an MOI of 3, harvested at 18 h postinfection, and immunoprecipitated with antibody to Us3. The immunoprecipitates were divided into two aliquots. One aliquot was incubated in kinase buffer containing [γ-32P]ATP and MBP-UL34, separated on a denaturing gel, transferred to a nitrocellulose membrane, stained with Ponceau S (upper panel), and analyzed by autoradiography (middle panel). The other was separated on a denaturing gel, transferred to a nitrocellulose membrane, and subjected to immunoblotting with the anti-Us3 antibody (lower panel). (B) MBP-UL34 incubated with the immunoprecipitates prepared for panel A was either mock treated (lane 1) or treated with λ-PPase (lane 2), separated on a denaturing gel, transferred to a nitrocellulose membrane, stained with Ponceau-S (upper panel), and analyzed by autoradiography (middle panel). Immunoprecipitates prepared for panel A were also separated on a denaturing gel, transferred to a nitrocellulose membrane, and subjected to immunoblotting with anti-Us3 antibody (lower panel).