Figure 7
Signal transduction and induced apoptosis are unchanged in (K395A/3F)Syk-infected cells despite constitutive Syk kinase activity (A) A vector-infected control (circle) and a (K395A/3F)Syk-infected clone with constitutive Syk activity, 2.1.2 (square), were plated at 1 × 105 cells/ml and 10 μg anti-IgM (closed symbols) or anti-ova (open symbols) added 24 hrs later on day 0. Samples were harvested on the indicated days, stained with Trypan blue and viable cell counts were determined. (B) The responses of a vector control clone (upper row) and (K395A/3F)Syk-infected clone 2.1.2 (lower row) to BCR signaling were analyzed. 2 × 105 cells were incubated with 15 μg of a polyclonal, goat anti-IgM antibody or 15 μg of a species-matched control antibody (control Ig) for 24 hours. Samples were harvested, stained with 7-AAD and Hoechst 33342, and analyzed by flow cytometry. In the dot plot analysis of the 7-AAD (x-axis) and Hoechst (y-axis) fluorescence, the apoptotic population is circled. The results presented are representative of three independent experiments with similar results. (C) Constitutive Syk activity does not give rise to a detectable increase in intracellular protein tyrosine phosphorylation. Cell lysates were prepared from unstimulated (no antibody (-) or anti-ova (C)) and stimulated (+; 15 μg anti-IgM for 0.5 minutes) BCL1.3B3, a vector-infected control, and (K395A/3F)Syk-infected clones 2.1.2 and 2.4.2 and analyzed by anti-PY immunoblotting. (D) PLCγ2 is not tyrosine phosphorylated in (K395A/3F)Syk-infected clones with constitutive Syk activity. PLCγ2 was immunoprecipitated from an unstimulated (-) and stimulated (+, 15 μg anti-IgM for 0.5 minutes) vector-infected control clone and (K395A/3F)Syk-infected clones 2.1.2, and 2.4.2. The immunoprecipitates were resolved by SDS-PAGE and analyzed by anti-PY immunoblotting (top panel). The blot was stripped and reprobed with an anti-PLCγ2 antibody (bottom panel). Anti-ova was used as an immunoprecipitation control (lanes 3, 6, and 9). The arrows mark the migration position of PLCγ2.