Immunoblotting of ALPK1. A. HEK293 or THP1 cell extracts (20 μg protein) were subjected to SDS-PAGE on 6% polyacrylamide gels and immunoblotted with 1.0 μg/ml of the ALPK1 antibody raised against amino acid residues 395-824 of ALPK1. The antibody was diluted in B. Same as A, except that the HEK293 cells were transfected with an ALPK1 siRNA or control siRNA prior to cell lysis. After siRNA knock-down of ALPK1, the 140 kDa band could no longer be detected by the antibody showing that the 140 kDA band was indeed ALPK1.
Immunprecipitation of ALPK1 in cell extracts. Increasing amounts of the antibody raised against amino acid residues 395-823 of ALPK1 or a control IgG were used to immunoprecipitate ALPK1 from 0.5 mg of THP1 cell extract protein in a total volume of 0.5 ml. After immunoprecipitation, the ALPK1 was released from the antibody in SDS, subjected to SDS-PAGE and immunoblotted using the same antibiody (first 6 lanes from the left). The supernatant obtained after immunoprecipitation was analysed in a similar manner (lanes 7-12 from the left) to estimate the proportion of ALPK1 that had been immunoprecipitated.