The expression and tyrosine phosphorylation of NTAL and LAT following FcϵRI aggregation in human mast cells. (A-B) Whole cell lysates (100 000 cell equivalents) of U937 cells (U), RBL 2H3 cells (R), mouse BMMCs (M), and HuMCs (H) were probed with anti-NTAL and anti-LAT monoclonal (m) (NAP 08) and polyclonal (p) antibodies. Comigration of (C) NTAL with pp30 in HuMCs and an equivalent tyrosine-phosphorylated protein in mouse BMMCs and (D) LAT with pp38 in HuMCs and in mouse BMMCs. HuMCs and BMMCs were triggered as described in “Materials and methods” and probed with an antiphosphotyrosine mAb (pY). Extracts as in panels A-B were probed with either an anti-NTAL pAb or anti-LAT mAb (NAP-08), and the gels were then aligned based on the migration of standard molecular-weight markers. (E-F) HuMCs were sensitized and triggered with NP-BSA (100 ng/mL) for 30 seconds (T), and then the phosphorylation was compared with nonactivated cells (N). The lysates were then probed with the indicated antibodies. The proteins in panels A-D were extracted under denaturing conditions and in panels E-F, under the conditions for immunoprecipitation as described in “Materials and methods.” IP indicates immunoprecipitation. The data are representative of n = 2 to 3.