Upregulation of Cortactin Expression During the Maturation of Megakaryocytes by Xi Zhan, Christian C. Haudenschild, Yangson Ni, Elizabeth Smith, and Cai Huang Blood Volume 89(2):457-464 January 15, 1997 ©1997 by American Society of Hematology

Polyclonal anticortactin antibody detects specifically cortactin proteins in murine fibroblasts. Polyclonal anticortactin antibody detects specifically cortactin proteins in murine fibroblasts. Murine fibroblasts (1 × 106 3T3 cells) were directly lysed in 300 μL of the SDS sample buffer. Ten microliters (lane 1) and 20 μL (lane 2) of the lysate were analyzed by Western blotting using purified polyclonal anticortactin antibody (C001) at the concentration of 1 μg/mL. Xi Zhan et al. Blood 1997;89:457-464 ©1997 by American Society of Hematology

Immunohistochemical analysis of cortactin expression in megakaryocytes. Immunohistochemical analysis of cortactin expression in megakaryocytes. Mature megakaryocytes stain positively in bone marrow, spleen, and liver. Platelets that cover the de-endothelialized intima (arrow) stain positively as well. (A) Bone marrow (original magnification × 160); (B) spleen (original magnification × 20); (C) liver of a mouse embryo (12.5 days, original magnification × 30); (D) a balloon-injured rat carotid artery (original magnification × 120) 24 hours after injury. Xi Zhan et al. Blood 1997;89:457-464 ©1997 by American Society of Hematology

Upregulation of cortactin expression in CMK cells in response to differentiation stimulus. Upregulation of cortactin expression in CMK cells in response to differentiation stimulus. (A) CMK cells were treated with PMA (10 nmol/L) for the time period as indicated, lysed, and immunoprecipitated with 4F11. The precipitates were analyzed in an immunoblotting using C001 polyclonal antibody. The detail procedure was described in the Materials and Methods. The data shown here are representative of four experiments. (B) CMK and CMK11-5 cells at an exponential growth stage in serum were analyzed for cortactin expression by immunoblotting analysis. The data are representative of two experiments, showing much more of cortactin proteins in the highly differentiated CMK subclone, CMK11-5. (C) Expression of mRNAs for cortactin and HS1 in PMA-treated CMK cells. The Northern blotting analysis was performed as described in the Materials and Methods. The intensities of mRNA for cortactin and HS1 were normalized with the ribosomal RNA 28S and plotted after a densitometry analysis. The autoradiography of the Northern blot is shown in the inset. The cortactin RNA is shown as a single 3.5-kb transcript, and HS1 RNA is shown as a single 2.0-kb band. The panels on the bottom show ethidium bromide staining of ribosomal RNA in agarose gel as the loading control. The results are representative of three experiments. Xi Zhan et al. Blood 1997;89:457-464 ©1997 by American Society of Hematology

Tyrosine phosphorylation of cortactin in CMK cells stimulated with PMA Tyrosine phosphorylation of cortactin in CMK cells stimulated with PMA. (A) CMK cells were treated with PMA for the time periods as indicated, lysed, and subject to immunoprecipitation with the polyclonal antibody 2719 against a cortactin-specific peptide.2... Tyrosine phosphorylation of cortactin in CMK cells stimulated with PMA. (A) CMK cells were treated with PMA for the time periods as indicated, lysed, and subject to immunoprecipitation with the polyclonal antibody 2719 against a cortactin-specific peptide.23 The immunoprecipitates were fractionated with an SDS-PAGE (7.5%) and immunoblotted with an antiphosphotyrosine antibody (anti–p-Tyr). (B) The membrane used above was stripped and reblotted with 4F11 monoclonal antibody. (C) CMK cells stimulated with PMA in parallel were lysed directly with SDS-sample buffer and analyzed for c-Src–related kinases using c-Src-2 antibody. Xi Zhan et al. Blood 1997;89:457-464 ©1997 by American Society of Hematology

Western blotting analysis of pp60c-src expression in PMA-treated CMK cells. Western blotting analysis of pp60c-src expression in PMA-treated CMK cells. CMK cells were treated with PMA. At the time indicated, 5 × 105 cells were harvested and lysed in SDS sample buffer. The presence of c-Src–related proteins in the lysates was analyzed using a purified c-Src polyclonal antibody (c-Src-2; Santa Cruz Biotechnology Inc). The results shown here are representative of three experiments. Xi Zhan et al. Blood 1997;89:457-464 ©1997 by American Society of Hematology

Regulation of cortactin expression by hematopoieticfactors in bone marrow. Regulation of cortactin expression by hematopoieticfactors in bone marrow. Bone marrow-derived cells were prepared and cultured for 4 days as described in the Materials and Methods. (A) Fresh bone marrow cells; (B) cells in 10% fetal bovine serum; (C) cells in Tpo (200 U/mL); (D) cells in IL-3 (3 ng/mL); (E) cells in IL-11 (50 ng/mL); and (F ) cells in Tpo plus IL-11. The results shown here are representative of two experiments. Xi Zhan et al. Blood 1997;89:457-464 ©1997 by American Society of Hematology