Fig. 2

Glutaminase C activity is elevated in NSCLC and regulated by phosphorylation. a Mitochondrial fractions were isolated from equivalent numbers of NSCLC cells and HBE cells, and then the glutaminase activity was determined. Data represent the average of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001. b H1299 cells were transiently transfected with pcDNA3.1-V5-GAC and then immunoprecipitated using an anti-V5 antibody. The samples were treated with alkaline phosphatase or left untreated for 1 h at 37 °C. Glutaminase activity was then assayed. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001 (AP alkaline phosphatase) (top figure). The GAC expression levels were checked by western blotting using an anti-V5 antibody (bottom figure). c HBE cells transiently transfected with V5-GAC were lysed and extracts subjected to isoelectric focusing; H1299 cells transiently transfected with V5-GAC were lysed and subjected to alkaline phosphatase or control treatment prior to isoelectric focusing. All the above experiments were followed by western blotting using an anti-V5 antibody. d The GAC phosphorylation sites in H1299 cells but not in HBE cells were identified by mass spectrometry. # represents a phosphorylation site. e H1299 cells transiently transfected with V5-GAC wild-type (GAC-WT) or GAC mutants (T112A, T188A, S274A, T278A, S314A, and S511A), were immunoprecipated using the anti-V5 antibody and then the glutaminase assay was performed. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001 (top figure). The GAC expression levels of wild-type and mutants were checked by western blotting with the anti-V5 antibody (bottom figure). f H1299 cells transiently transfected with V5-GAC wild-type (GAC-WT) or GAC mutant (GAC-S314D) were immunoprecipated using the anti-V5 antibody before glutaminase assay. Data represent the average of three independent experiments (mean ± SD). *P < 0.05 (top figure). The GAC expression levels of wild-type and mutant were checked by western blotting using the anti-V5 antibody (bottom figure). g The expression of total and phosphorylated GAC at serine 314 in NSCLC cell lines and HBE cells was determined by western blotting using anti-GAC and anti-GAC-pS314 antibodies