Btk is involved in GM-CSF–mediated signaling and is necessary for the sufficient expression of transcription factors important for efficient granulopoiesis. (A) MACS-sorted CD11b⁺ cells from the bone marrow of wild-type (wt) mice were treated for the indicated time with GM-CSF. Cells were lysed and analyzed by immunoblotting for the phosphorylation of Btk at Y551 (Btk pY551). The membrane was then stripped and analyzed for the amount of total Btk protein loaded onto the gel (Btk). (B-C) Erythrocytes-depleted bone marrow cells of wild-type (wt) or Btk-deficient (Btk-ko) mice or Xid mice (xid) were treated for the indicated time with GM-CSF. Cells were lysed and analyzed by immunoblotting for the phosphorylation of Stat3 (pStat3), Akt (pAkt), PI3K (pPI3Kp85), and GSK-3β (pGSK-3β), as well as for the total amounts of Stat3, Akt, and Erk2 proteins used for analyses. (D) CMP, GMP, and MEP were isolated from the bone marrow of wild-type (wt) and Btk-deficient (Btk-ko) mice by FACS. RNA was prepared and analyzed for the mRNA expression of Btk (Btk), C/EBPα (Cebpa), PU.1 (Sfpi1), GATA1 (Gata1), and target genes like GM-CSF-Rα (Csf2ra), G-CSF-R (Csf3r), elastase (Elane), and myeloperoxidase (Mpo) by real-time PCR relative to the expression of Hprt. (E) Total bone marrow cells were isolated from wild-type (wt) and Btk-deficient (Btk-ko) mice. RNA was prepared and analyzed for the mRNA expression of C/EBPα (Cebpa) relative to the expression of Gapdh. (F) Bone marrow cells of wild-type and Btk-deficient mice were lysed and analyzed for the expression of C/EBPα. As a control for the genotype of mice used, a Western blot was performed using an anti-Btk antibody. Probing of the membrane with an anti Erk2-antibody served as loading control. (G) Total bone marrow cells were isolated from wild-type (wt) and Btk-deficient (Btk-ko) mice. RNA was prepared and analyzed for the mRNA expression of PU.1 (Sfpi1), M-CSF-R (Csf1r), and GM-CSF-Rα (Csf2ra) relative to the expression of Gapdh. (H) Bone marrow-derived neutrophils were isolated from wild-type (wt) and Btk-deficient (Btk-ko) mice and analyzed for the expression of C/EBPβ (Cebpb) relative to the expression of β-actin (Actb). Data presented are mean values (± SD). *P ≤ 0.05, **P ≤ 0.005. n represents the number of biological replicates. (B,F) Vertical lines have been inserted to indicate a repositioned gel lane.