Figure 4: Adenylate kinase hCINAP promotes LDHA Y10 phosphorylation catalysed by FGFR1.

(a) Western blot analysis of LDHA Y10 phosphorylation levels in SW480 cells transfected as indicated. See also hCINAP-enhanced LDHA tetramerization in Supplementary Fig. 4. (b) Immunoblot analysis of LDHA Y10 phosphorylation catalysed by FGFR1 in vitro with or without hCINAP. Y10F LDHA served as a negative control. (c) Western blot analysis of LDHA Y10 phosphorylation in SW480 cells transfected with the indicated plasmids and treated with FGFR1 inhibitor BGJ398 (10 nM) or DMSO. (d,e) The effects of wild-type hCINAP and H79G mutant hCINAP on FGFR1-catalysed LDHA Y10 phosphorylation in vitro (d) and in SW480 cells expressing the indicated plasmids (e). (f) The effect of AP5A on LDHA Y10 phosphorylation in FGFR1 kinase assay in vitro. (g) In vitro FGFR1 kinase assay with various combinations of protein and nucleotides as indicated. (h) ATP levels were measured by a luciferase reporter system in assays with the indicated combinations of protein and nucleotides. The results are expressed as mean±s.e.m. of three independent experiments. Asterisks indicate significant differences (***P<0.001 versus second column) determined by Student's t-test.