HEK 293 cells were transfected with constructs for Flag tagged wildtype and kinase dead (K38A) IKK epsilon. The transfected IKK epsilon was immunoprecipitated from cell extracts using anti-Flag M2 agarose (Sigma). The resulting immunoprecipitate was subjected to SDS-Page followed by immunoblotting with anti-FLAG (Sigma) and anti-IKK phospho Ser 172 antibodies (in the presence of non-phosphorylated or phosphorylated peptide). Binding of IKK epsilon phospho Ser 172 antibody was detected using rabbit peroxidase conjugated anti-sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).

RAW264.7 cells were unstimulated (-) or stimulated (+) with 100 ng/ml LPS for 30 mins. 1 mg of cellular extract was subjected to immunoprecipitation using 10 ug anti-IKK epsilon phospho Ser 172 in the presence of 100 ug of non-phosphorylated peptide. The resulting immunoprecipitate was subjected to SDS-Page followed by immunoblotting with anti-TBK1 phospho Ser 172, anti-IKK phospho Ser 172, anti-TBK1 (CST) and anti- IKK epsilon (Sigma). Binding of TBK1 and IKK epsilon phospho specific antibody was detected using rabbit peroxidase conjugated anti-sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).