Fig. 8.
Phosphorylation of serine 170 or serine 173 does not affect early BCR signaling events. A, DT40 B cells expressing either wild-type, S173A, or S170A mutant GFP-SLP-65 (as described in the legend to Fig. 7A) were left untreated (0 min; lanes 1, 5, and 9) or stimulated for 2 min (lanes 2, 6, and 10), 10 min (lanes 3, 7, and 11), or 20 min (lanes 4, 8, and 12) through their BCR. Induction of global protein tyrosine phosphorylation was monitored by anti-phosphotyrosine immunoblotting of cleared cellular lysates. B, cells described in A were loaded with the ratiometric Ca2± chelating dye Indo-1 and subsequently stimulated through their BCR. Induction of intracellular Ca2± mobilization was recorded by flow cytometry calculating the ratio between Indo-1 blue (405 nm) and Indo-1 violet (530 nm) for normalizing the results according to the loading efficiency. Curves represent intracellular Ca2± concentrations of stimulated cells expressing no SLP-65 (gray), wild-type GFP-SLP-65 (blue), GFP-SLP-S170A (red), and GFP-SLP-65-S173A (green).