Fig. 7.

Expression of wild-type SLP-65 as well as its S170A- and S173A mutant forms with an N-terminal GFP moiety. A, SLP-65-deficient DT40 B cells were subjected to retroviral transduction inducing stable expression of a GFP fusion protein encompassing either wild-type SLP-65 (GFP-SLP-65) or mutant SLP-65 variants in which either serine 170 or serine 173 was replaced by alanine (GFP-SLP-65-S170A or -S173A). GFP-positive cells were isolated by fluorescence-activated cell sorting to achieve populations expressing equal amounts of GFP-SLP-65 (left panel). Equal BCR expression of the obtained cell populations was confirmed by flow cytometry using Cy-5-labeled anti-IgM antibodies (right panel). B, cells described in A were left untreated (0 min; lanes 1, 6, and 11) or BCR-stimulated for indicated times in minutes (lanes 2–5, 7–10, 12–15). From cleared cellular lysates GFP-SLP-65 proteins were purified by anti-GFP immunoprecipitation, and obtained proteins were analyzed by immunoblotting with anti-Ser(P)-170, anti-Tyr(P), and anti-chicken SLP-65 antibodies (upper, middle, and lower panel, respectively).