Figure 1.
mAb specificity for intracellular detection of individual Btk tyrosine phosphorylation sites. Vaccinia viral stocks for Btk and Lyn proteins were produced and titered as described (19). Jurkat T cells (2 × 106 cells per ml) were infected by incubation with vaccinia virus stocks (multiplicity of infection = 5) for 1 hr at 4°C and grown for 8 hr at 37°C. Cells expressing Lyn wild type and/or Btk (wild type, Y551F, or Y223F) were fixed, permeabilized, and stained with antibodies (anti-Btk NT and either 551PYmAb or 223PYmAb) as indicated. The primary antibodies were detected with fluorescein isothiocyanate-labeled donkey anti-rabbit IgG and R-phycoerythrin-labeled donkey anti-mouse total IgG. Two-color fluorescence analysis was performed for each sample.