mTORC1 represses IL-9 transcription through inhibition of NF-κB signaling. (A) Western blot analysis of p-p65NF-κB (S653) and p-IKK-β (S180/181) in TSC1-deficient osteoblast. (B) Nuclear and cytosol localization of p65 in TSC1-deficient osteoblasts. Histone H1 and β-actin were used as nuclear and cytosolic protein markers, respectively. (C) EMSA of DNA binding activity of NF-κB to Il9 gene promoter in the TSC1 deletion or control osteoblasts. (D) Western blot analysis of p-p65NF-κB (S653) and p-IKK-β (S180/181) in Raptor-deficient osteoblast. (E) Nuclear and cytosolic localization of p65 in Raptor-deficient osteoblasts. Histone H1 and β-actin were used as nuclear and cytosol protein markers, respectively. (F) EMSA of DNA binding activity of NF-κB to Il9 gene promoter in the Raptor deletion or control osteoblasts. (G) IL-9 mRNA levels in Raptor-deficient osteoblasts (ROs) treated with NF-κB inhibitor (PDTC) or PI3-K/Akt inhibitor (LY294002), as detected by quantitative polymerase chain reaction (n = 3, 1-way ANOVA). (H) Western blot analysis of p-p65NF-κB (S653) and p-IKK-β (S180/181) in osteoblast (OB) cells treated with 50 nM rapamycin. (I) Quantitative polymerase chain reaction analysis of IL-9 mRNA levels in OBs treated with rapamycin (50 nM) and PDTC (50 μM), or LY294002 (50 μM) (n = 3, 1-way ANOVA). Data are representative of 3 independent experiments and are represented as mean ± SD. **P < .01.