Fig. 4.
Induction of GCN pathway in CD98hc ablation. (A) GCN and mTORC1 signaling in CD98hcflox/floxLysM-cre peritoneal macrophages. Peritoneal macrophages were cultured for 3 days (targeting phase) after treatment with M-CSF and RANKL. Cell lysates were prepared and probed by immunoblotting with the antibodies listed in the Materials and Methods section. “W” or “KO” means macrophages obtained from control or CD98hc knockout mice, respectively. (B) The experiment was performed with 6 mice per group. The staining density were normalized. Data are representative of means+SEM (**p<0.01). (C) Peritoneal macrophages were cultured for 24 h (amplifying phase) after treatment with M-CSF and RANKL. mRNA transcripts were amalyzed with GeneChip analysis. Left panel: mRNA expression level at 24 h; right panel: the fold increases in the mRNA expression level compared with that in unstimulated cells (control). ASNS: asparagine synthetase, GCN2: general control nonderepressible 2, eukaryotic translation initiation factor 2 alpha kinase 4, TRAP: Tartrate-resistant acid phosphatase, NFATc1: nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1, RANK: Receptor activator of nuclear factor kappa-B, Ctsk: Cathepsin K, Mmp9: Matrix metallopeptidase 9, Itgb3: integrin beta 3, Calcr: calcitonin receptor, Tm7sf4 DC-STAMP: Dendritic Cells (DC)-Specific Transmembrane Protein, Acp5: Acid Phosphatase 5.