Figure 3From: Activation of the Syk tyrosine kinase is insufficient for downstream signal transduction in B lymphocytesExpression of the (K395A/3F)Syk molecule is associated with constitutive Syk kinase activity (A) The amino acid changes introduced into wildtype Syk to create the (K395A/3F) Syk mutant include the substitution of lysine at amino acid 395 with alanine and the substitution of three tyrosines at amino acid positions 624–6 in the carboxy-terminus with phenylalanines. (B) The strategy for simultaneously detecting both the (K395A/3F)Syk and endogenous Syk mRNA by RT-PCR involves the use of three primers. In this case, Primer 3 is complementary to sequences in both the transgene and endogenous syk gene cDNAs, while Primers 1 and 2 are specific for sequences in the transgene and endogenous syk gene cDNA, respectively. The PCR products produced with Primers 1 and 3 from the transgene cDNA template are larger than the PCR products produced with Primers 2 and 3 from the endogenous gene cDNA template. The size difference can be assessed following separation by agarose gel electrophoresis. (C) mRNA expression levels of the (K395A/3F)Syk transgene (T) and endogenous (E) Syk gene in BCL1.3B3 (lane 2), a clone infected with the empty retroviral vector (lane 3), and (K395A/3F)Syk mutant clones (lanes 4–10) were determined using the RT-PCR strategy described above. All samples were prepared with (+R.T., upper panel) or without (-R.T., lower panel) reverse transcriptase to control for amplification of contaminating genomic DNA. (D) Syk activity was evaluated following immunoprecipitation with anti-Syk antibodies from unstimulated (-) or stimulated (+; 15 μg of anti-IgM for 30 seconds) BCL1.3B3 or (K395A/3F)Syk-containing clones (2.1.6, 2.4.2, 2.1.2, and 2.1.7) by the ICKA. Anti-ova was used as an immunoprecipitation control (C; lanes 3, 6, 9, 12, and 15). (E) BCL1.3B3, cells infected with the MSCV2.1 expression vector alone (vector control) and (K395A/3F)Syk-infected clones 2.1.2 and 2.1.6, were incubated with or without 15 μg of anti-IgM or anti-ova for the indicated times and Syk activity evaluated by the ICKA (lanes 1–6). Anti-ova-coated beads were used as an immunoprecipitation control (C; lane 7). (F) Syk was immunoprecipitated from unstimulated (no antibody (-) or anti-ova (C)) or stimulated (+; 15 μg anti-IgM for 0.5 minutes) BCL1.3B3, a vector control clone, and (K395A/3F)Syk clones 2.1.2 and 2.4.2. The immunoprecipitates were resolved by SDS-PAGE and evaluated by anti-PY immunoblotting (upper panel). The blot was stripped and reprobed with an anti-Syk antibody (lower panel). Our data suggests that the binding activity of the antibody used for immunoprecipitation is partly influenced by the phosphorylation status of Syk; although phosphorylation is not required for antibody binding, the amount of Syk present in the immunoblot reflects the level of phosphorylation of the protein to some degree.Back to article page