Pecy7 anti ckit

BM cells were collected from tibias and femurs and stained with anti-c-kit PE-Cy7 (BD Biosciences), anti-Sca-1 APC-Cy7 (BD Biosciences), anti-lineage V450 (BD Biosciences), anti-CD48 FITC (Biolegend), anti-CD127 FITC (BD Biosciences), anti-CD41 Alexa Fluor 488 (Biolegend), anti-CD34 APC (BD Biosciences), anti-CD135 PE (BD Biosciences), anti-CD16/32 PerCP (BD Biosciences) and anti-CD150 Alexa Fluor 647 (Biolegend). BM HSCs (CD150⁺CD48^-Lineage^-Sca-1⁺c-Kit⁺), MPPs (CD150^-CD48^-Lineage^-Sca-1⁺c-Kit⁺), CMPs (CD34⁺CD16/32^lowCD127^-Lineage^-Sca-1^-c-Kit⁺), GMPs (CD34⁺CD16/32^highCD127^-Lineage^-Sca-1^-c-Kit⁺), MEPs (CD34^-CD16/32^-/lowCD127^-Lineage^-Sca-1^-c-Kit⁺), MkPs (CD150⁺CD41⁺Lineage^-Sca-1^-c-Kit⁺) and EPs (CD71⁺Ter119⁺) were stained with the indicated cell surface marker antibodies. For intracellular phosphoprotein analysis, BM cells were flushed into serum-free PBS. BM cells were stained with surface marker antibodies, then washed with PBS and fixed for 15 minutes at 4°C, followed by incubation with BD c ytoperm buffer for 30 minutes at room temperature. Rabbit anti-pS6K (Thr389) (Cell Signaling Technology) or anti-pAkt PE (Cell Signaling Technology) were added at 1:50 dilution for 30 minutes. For pS6K staining, cells were incubated with anti-rabbit IgG PE (Cell Signaling Technology) at 1:500 for 30 minutes. Flow cytometric analysis was performed on a FACS CANTO II (BD Biosciences).