FIG. 6 .
6Photomicrographs of Cx43 immunoreactivity in osteoblastic cells. Cells plated at 50,000 cells/cm 2 and grown for 48 h were fixed, permeabilized, and incubated with a polyclonal antibody to rat Cx43. A fluorescein-conjugated secondary antibody was used for visualization under a broadband ultraviolet light. ROS cells (A) exhibit a punctate staining pattern that represents gap junctions composed of Cx43 located primarily in the plasma membrane between adjacent cells. Protein reserves of Cx43 are those spots located perinuclearly. RCϫ16 (B) show no immunoreactivity at the cell surface and little within the cytoplasm. These experiments were repeated four to seven times for each cell line examined. FIG. 5. Western blot analysis of connexin43 protein in osteoblastic cells. Equivalent amounts of total cell extract enriched for membrane proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed using a polyclonal antibody to rat Cx43. Lack of immunoreactivity for nonphosphorylated Cx43 (Cx43-NP) and all phosphorylated forms (Cx43-P1 and Cx43-P2) was observed for RCx4 (lane 3). RCx16 demonstrated greatly reduced immunoreacitivity for all Cx43 forms, whereas ROS (lane 1) and bG (lane 2) showed abundant immunoreactivity for all forms of Cx43 protein. These **data** are typical of three similar experiments.
