Fig. 1

Cyclin G2 reduced Y10 phosphorylation of LDHA catalyzed by FGFR1. a-b Western blotting of whole-cell lysates (WCL) from U87 and U251 cells transfected with scrambled non-targeted (siNC) or cyclin G2 siRNA (siCCNG2) using corresponding antibodies, followed by IP with anti-FGFR1, probed with anti-LDHA. Mouse IgG was used as a negative IP control. GAPDH was used as a loading control for western blotting analysis. Bands were quantified using the Image J. c-d Proximity ligation assay (PLA) of FGFR1 and LDHA in U87 and U251 cells transfected with siNC or siCCNG2. Red spots indicate FGFR1/LDHA interaction and DAPI was used as a nuclear marker. Original magnification, 1000×. Scale bar, 50 μm. The histogram quantifies the number of red spots per cell (n = 50 cells in two technical replicates). The results are representative of three independent experiments. e-f After starvation in serum free DMEM media for 24 h, Y10 phosphorylation of LDHA in U87 and U251 cells was detected by western blotting following bFGF stimulation (10 ng/mL, 3 h). g-h Western blotting was performed to examine the effect of cyclin G2 on Y10 phosphorylation of LDHA in the presence or absence of BGJ398. i In vitro kinase assay and western blotting was performed to examine Y10 phosphorylation of recombinant LDHA WT or LDHA Y10F by FGFR1 in the presence or absence of cyclin G2. Results shown are representative of two independent experiments. Data from three independent experiments are presented as the mean ± s.d. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ns, not significant