Lck kinase activity is not inhibited by RosA.
(A) The effect of RosA on TCR-induced activation of Lck kinase activity. Lck was immunoprecipitated by anti-Lck mAb (5 μg/mL) from Jurkat cell lysates and analyzed for its ability to phosphorylate enolase (top panel). Lck kinase assay was performed as described in “Materials and methods.” Autophosphorylation status (middle panel) and equivalent immunoprecipitation of Lck (bottom panel) was confirmed by Western blotting using antiphosphotyrosine antibody and anti-Lck mAb (1 μg/mL), respectively. (B) The effect of RosA on OKT3/4-induced Lck activation. After preincubation with or without RosA, Jurkat cells were harvested and activated with OKT3/4 for 2 minutes. Jurkat cell lysates (2 × 106 cells per lane) were subjected to 12% SDS-PAGE, transferred to the nitrocellulose membrane, and blotted with anti-Lck mAb (1 μg/mL). Lck isoforms p56 and p59 are indicated by arrows. Results are representative of 2 separate experiments.
