Fig. 3

PIM kinases phosphorylate LKB1 in vitro and LKB1 interacts with PIM1 in cells. a Radioactive in vitro kinase assays were performed by incubating GST, GST-PIMs and/or GST-LKB1 in the presence of ³²P-ATP. Phosphorylation signals were analysed by autoradiography (upper panel), while protein loading was visualised by Page Blue staining (lower panel). Shown is a representative image out of two repeated experiments. b Similar non-radioactive in vitro kinase assays were analysed by Western blotting with phospho-AKT substrate (PAS) antibody (upper panel), while protein loading was visualised by stain-free technology (lower panel). c Shown are the PIM kinase consensus phosphorylation motif, the PAS antibody recognition site, and the LKB1 sequences around residues Ser334 and Ser428. d Radioactive in vitro kinase assays (n = 2) were performed by incubating GST-PIM1 with His-tagged wild-type (WT) or mutant (S334A or S428A) LKB1. e Similar non-radioactive in vitro kinase assays (n = 2) were performed using GST-tagged PIM1 and LKB1 (WT or S334A) proteins. f MCF7 and PC3 cells were transiently transfected with FLAG-tagged LKB1 and His or His-tagged PIM1 plasmids. After 24 h, 10% of cell lysates were stained with FLAG antibody (input), while the rest was immunoprecipitated with FLAG M2 affinity gel and stained with His antibody (IP: FLAG). g MCF7 and PC3 cells were transiently transfected with GFP, GFP-tagged LKB1, RFP and/or RFP-tagged PIM1 plasmids. After 24 h, cells were fixed and analysed by fluorescence-lifetime imaging microscopy (FLIM). Shown are representative FLIM images as well as graphs (average value ± SD), where numbers of counted cells have been indicated