1. by Sansana Sawasdikosol, Kristin M. Russo, and Steven J. Burakoff
Hematopoietic progenitor kinase 1 (HPK1) negatively regulates prostaglandin E2–induced fos gene transcription
by Sansana Sawasdikosol, Kristin M. Russo, and Steven J. Burakoff
Blood
Volume 101(9):
May 1, 2003
©2003 by American Society of Hematology

2. Physiological concentrations of PGE2-induced HPK1 kinase activity
Physiological concentrations of PGE2-induced HPK1 kinase activity.HA-HPK1 was immunoprecipitated from lysates derived from Jurkat T cells transfected with HA-HPK1.
Physiological concentrations of PGE2-induced HPK1 kinase activity.HA-HPK1 was immunoprecipitated from lysates derived from Jurkat T cells transfected with HA-HPK1. Cells were exposed to varying concentrations of PGE2 for 2 minutes at 37°C prior to lysis and immunoprecipitation. In vitro immune complex HPK1 kinase assays (IVK) were performed in the presence of exogenous substrate, 5 μg histone H2A. (A) 32P-incorporated histone H2A. (B)32P-incorporated auto-transphosphorylated HA-HPK1. (C) Western blot of immunoprecipitated HPK1 from panel B, using the anti-HPK1 no. 7 antibody. (D) In vitro immune complex HPK1 kinase assays were performed on lysates of PGE2-stimulated (10 nM) transfectants expressing wild-type or a catalytically inactive mutant (K46E) of HPK1. The autoradiographic bands depict32P-incorporated histone H2A. (E)32P-incorporated autophosphorylated/transphosphorylated wild-type and (K46E) mutant HPK1. (F) Western blot of immunoprecipitated HPK1 from panel E, using the anti-HPK1 no. 7 antibody. (G) In vitro immune complex kinase assays performed with endogenous HPK1 immunoprecipitated from nontreated cells or from cells treated with 10 nM PGE2. (H) Western blot of immunoprecipitated HPK1 from panel G, using the anti-HPK1 no. 7 antibody. Lane numbers are indicated at the bottom of all lanes. Data in all figures are representative of at least 3 independent experiments.
Sansana Sawasdikosol et al. Blood 2003;101:
©2003 by American Society of Hematology

3. HPK1 inhibits PGE2-induced fos promoter activity.
HPK1 inhibits PGE2-induced fos promoter activity. (A) Fospromoter–regulated relative luciferase activity from cells transfected with empty vector or HPK1 constructs. Transfectants were left untreated (■) or were stimulated with 10 nM PGE2 (▪) for 6 hours prior to cell harvest. Duplicate transfectants were lysed, and the lysates were tested for luciferase activity. Relative light unit (RLU) values were normalized with the Renilla luciferase values. Error bars indicate standard deviations. This histogram depicts an experiment that represents the trend observed in 4 of 5 experiments. (B) Western blot analysis revealing the expression levels of the transfected HA-HPK1.
Sansana Sawasdikosol et al. Blood 2003;101:
©2003 by American Society of Hematology