Fig. 2. Purification of recombinant GST-PCTAIRE-1 and detection of PCTAIRE-1 kinase activity. Full-length PCTAIRE-1 was expressed as a GST-fusion protein in insect Sf-9 cells. The recombinant protein was affinity-purified on a glutathione-Sepharose 4B column as described in "Materials and Methods." Purified GST-PCTAIRE-1 (2 µg) was resolved on a 10% polyacrylamide gel in the presence of SDS and either
stained with Coomassie Blue (A) or analyzed by Western blotting with an antibody to PCTAIRE-1 (B) or GST (C). D, HA-tagged PCTAIRE-1 was ectopically expressed in HeLa cells, and kinase activity was determined using MBP as a substrate. Extracts from cells transfected with the empty vector (Lanes 1 and 2) or HA-tagged PCTAIRE-1 (Lane 3) were left untreated (Lane 1) or were immunoprecipitated with the 12CA5 antibody (Lanes 2 and 3). Samples were treated with protein G-Sepharose, and immunocomplexes were tested for kinase activity as described in "Materials and Methods." Proteins were resolved by SDS-PAGE followed by autoradiography. E, in gel kinase assay. Equal amounts of protein (200 µg) derived from the extracts of untreated and H2O2-treated Jurkat cells (Lanes 1 and 2) or HA-tagged PCTAIRE-1-expressing Hs68 (Lane 3), HeLa (Lane 4), and COS-7 cells (Lane 5) were immunoprecipitated with anti-ERK2 (Lanes 1 and 2) or anti-PCT-1 antibody (Lanes 35). An asterisk indicates the position of ERK2. Lane 6 contains affinity-purified GST-PCTAIRE-1 (15 µg). Proteins were separated on a 10% polyacrylamide gel containing 0.5 mg/ml MBP under denaturing conditions. Renaturation of the resolved proteins and determination of kinase activity were carried out as described in "Materials and Methods." F, purified GST-PCTAIRE-1 (5 µg) was examined for kinase activity on MBP before (Lane 1) or upon (Lane 2) a 2-h incubation on ice with 50 µg of fibroblast cell extract.