RosA does not interfere with the TCR-induced tyrosine phosphorylation of the TCR ζ-chain and ζ-chain–associated ZAP-70.

(A) The effect of RosA on phosphorylation status of ζ-chain and ζ-chain–associated ZAP-70. Jurkat cells were preincubated with or without RosA (30 μM) for 4 hours and stimulated for 3 minutes. Jurkat cells (5 × 10⁷) were then lysed, immunoprecipitated with anti-TCR ζ-chain mAb (5 μg/mL), and separated by 12% SDS-PAGE. The tyrosine phosphorylation status of the ζ-chain and ζ-chain–associated ZAP-70 was verified by Western blot using antiphosphotyrosine mAb (top panel). The same membrane was reprobed with anti-TCR ζ-chain mAb (1 μg/mL) (bottom panel) to confirm the equal immunoprecipitation of the ζ-chain. (B) The effect of RosA on the tyrosine phosphorylation of ZAP-70. ZAP-70 was immunoprecipitated using anti–ZAP-70 mAb (5 μg/mL) and probed with antiphosphotyrosine mAb (top panel). The same blot was stripped and reprobed with anti–ZAP-70 mAb (bottom panel) (1 μg/mL). Results represent one of 3 independent experiments.