Recombinant GST-IRF3 protein expressed in E. coli Rosetta strain was purified with glutathione sepharose and eluted from beads with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione). For in vitro kinase assays, vector, Flag-tagged BLK and its mutant plasmids were individually transfected into HEK293 cells for 24 h. These proteins were then purified with Protein G sepharose beads and co-incubated with purified GST-IRF3 (50 mg) in an equal volume of 2 × reaction buffer (100 mM Tris-HCl, 20 mM MgCl2, 1 mM Na3VO4, 4 mM DTT, pH 7.2), and 1 mM ATP was added prior to incubation for 1 h at 30 °C. The reaction was stopped by adding 1/5 volume of 6 × SDS loading buffer and boiled for 15 min at 95 °C before immunoblot analysis. For BLK autophosphorylation assays, vector, Flag-tagged BLK and its mutants plasmids were individually transfected into HEK293 cells for 24 h. These proteins were then purified with Protein G sepharose beads and incubated with 50 mL of 1 × reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM Na3VO4, 2 mM DTT, pH 7.2), and 1 mM ATP was added prior to incubation for 1 h at 30 °C. The reaction was stopped by adding 1/5 volume of 6 × SDS loading buffer and boiled for 15 min at 95 °C before immunoblot analysis.