The Related Adhesion Focal Tyrosine Kinase (RAFTK) Is Tyrosine Phosphorylated and Participates in Colony-Stimulating Factor-1/Macrophage Colony-Stimulating Factor Signaling in Monocyte-Macrophages by William C. Hatch, Ramesh K. Ganju, Dananagoud Hiregowdara, Shalom Avraham, and Jerome E. Groopman Blood Volume 91(10):3967-3973 May 15, 1998 ©1998 by American Society of Hematology

Tyrosine phosphorylation of RAFTK in THP1 monocytic cells and primary MMs. THP1 cells (20 × 106) were stimulated with either (A) 1,000 U/mL CSF-1/M-CSF or (B) 2 μg/mL LPS for the indicated time periods. Tyrosine phosphorylation of RAFTK in THP1 monocytic cells and primary MMs. THP1 cells (20 × 106) were stimulated with either (A) 1,000 U/mL CSF-1/M-CSF or (B) 2 μg/mL LPS for the indicated time periods. (C) MMs (20 × 106) were allowed to adhere and mature in culture for 7 to 14 days before their stimulation with 1,000 U/mL CSF-1/M-CSF. Cell lysates prepared in RIPA buffer were subjected to immunoprecipitation with anti-RAFTK antibody or normal rabbit serum as a control. Anti-RAFTK immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antiphosphotyrosine antibody (4G10) (top panel). The same blot was subjected to serial immunoblotting with anti-RAFTK antibody (bottom panel). TCL, total cell lysates; NRS, normal rabbit serum control. William C. Hatch et al. Blood 1998;91:3967-3973 ©1998 by American Society of Hematology

Association of RAFTK with PI-3 kinase in THP1 cells. Association of RAFTK with PI-3 kinase in THP1 cells. THP1 cells (20 × 106) were stimulated with either (A) CSF-1/M-CSF (1,000 U/mL) or (B) LPS (2 μg/mL) for the indicated time periods. Cell lysates prepared in RIPA buffer were subjected to immunoprecipitation with anti-RAFTK antibody. Anti-RAFTK immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti–PI-3 kinase antibody. TCL, total cell lysates. William C. Hatch et al. Blood 1998;91:3967-3973 ©1998 by American Society of Hematology

PI-3 kinase activity associates with RAFTK in THP1 cells. PI-3 kinase activity associates with RAFTK in THP1 cells. THP1 cells were stimulated with either 1,000 U/mL or 2 μg/mL LPS for 2 minutes and lysed in RIPA buffer without sodium deoxycholate. Lysates were immunoprecipitated with either anti-RAFTK, normal rabbit serum control, or anti–PI-3 kinase p85 antibody. Immune complexes were absorbed to sepharose-A beads for 3 hours, washed, and subjected to PI-3 kinase assay. Lipids were extracted using methanol:chloroform (1:1) and spotted on oxalate-impregnated silica gel TLC plates. Samples were subjected to ascending chromatography using a methanol:chloroform:water:ammonium hydroxide solvent system. TLC plates were dried and samples were visualized by autoradiography. William C. Hatch et al. Blood 1998;91:3967-3973 ©1998 by American Society of Hematology

Association of RAFTK with the c-fms receptor in THP1 cells. Association of RAFTK with the c-fms receptor in THP1 cells. THP1 cells (20 × 106) were stimulated with 1,000 U/mL CSF-1/M-CSF for the indicated time periods. Cell lysates prepared in RIPA buffer were subjected to immunoprecipitation with either the anti-c-fms antibody (A) or anti-RAFTK antibody (B). Immune complexes were absorbed onto sepharose-A beads, washed and resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with either anti-RAFTK (A) or anti–c-fmsantibody (B). NRS, normal rabbit serum control. William C. Hatch et al. Blood 1998;91:3967-3973 ©1998 by American Society of Hematology

Reduction of MAP Kinase activity by overexpression of a RAFTK dominant-negative kinase mutant. Reduction of MAP Kinase activity by overexpression of a RAFTK dominant-negative kinase mutant. THP1 cells were stably transfected with the RAFTKpcDNA vector control or with RAFTKm457 dominant-negative kinase mutant. THP1 transfectants (20 × 106) were stimulated with CSF-1/M-CSF (1,000 U/mL), and then cell lysates were prepared in RIPA buffer. Lysates were subjected to immunoprecipitation with anti-ERK1 and ERK2 antibodies. Immune complexes were absorbed with sepharose-A beads and then washed and subjected to in vitro kinase assay for 30 minutes. Samples were subjected to 15% SDS-PAGE and to autoradiography at −80°C. William C. Hatch et al. Blood 1998;91:3967-3973 ©1998 by American Society of Hematology