Fig. 1

CPs are phosphorylated by and co-expressed with PIM1 in prostate cancer. a Radioactive in vitro kinase assays were performed by incubating human GST-tagged wild-type (WT) or kinase-deficient (KD) PIM1 with murine Notch1 intracellular domain (N1ICD, a positive control) or with murine His-Capza1 or human His-CAPZA2 together with murine Capzb2. Shown are results from one representative experiment out of three assays. Relative phosphorylation signal intensities as compared to loaded amounts of protein are shown under the blots. bCAPZ mRNA levels expressed in human PC-3 prostate cancer cells were obtained from the IST Online™ database. c-d Pearson’s correlation coefficiencies were determined to PIM and CAPZA1 or CAPZB mRNA levels in human prostate cancer patient samples obtained from the Betastasis database. Shown are correlations (r²) along with significance (P) values. e Mass spectrometry (ms) of in vitro and in vivo phosphorylated CP samples as well as in silico analysis were used to predict PIM kinase target sites. In vitro kinase assays were performed with GST-tagged PIM1 and either wild-type (WT) CP or serine to alanine (SA) mutants, where two alpha sites (2X; S106, S126) or three beta sites (3X; S182, S192, S226) had been mutated. Shown are representative figures with relative signal intensities. f Proteins interacting with Flag-Capzb2 were analysed from lysates of transiently transfected PC-3 cells. Shown are examples of immunoprecipitation (IP), immunoblotting (IB) and and co-immunoprecipitation (CO-IP). Note that the PIM1 antibody non-specifically detects also other proteins, most likely immunoglobulins