Fig. 8

a Lineage and species specificity of the cell-surface A7 antigen expression: Western blotting and immunoprecipitation. Western blot analysis: Proteins extracted from cultured ROS17/2.8 CL#7 cells (day 4) were subjected to 10% SDS-PAGE and Western blotting was performed as described in Materials and Methods. Following electroblotting, membrane was incubated with isotype control IgG (lane 1) or A7 MAb (lane 2), respectively. Although no band was detected by the isotype control IgG, single protein band of 45 KDa was clearly detected by A7 MAb. b Lineage and species specificity of A7 antigen expression: Western blot analysis of cell lysates extracted from cell lines of several species were subjected to 10% SDS-PAGE. Following electroblotting, membrane was incubated with isotype control IgG (upper panel), A7 MAb (middle panel) or anti-β-tubulin antibody (lower panel). Rat cell lines: Osteoblastic ROS17/2.8 CL#7 cells, osteoblastic UMR106 cells, myoblast L8 cells and alveolar macrophage NR8383 cells; Murine cell lines: Osteoblastic MC3T3-E1 cells, osteoclast precursor RAW-D cells ± RANKL (50 ng/ml) and human cell lines: Osteosarcoma Saos-2 cells and MG-63 cells. c Biotinylation of cell-surface proteins in ROS17/2.8 cell clone. Cell-surface proteins of ROS17/2.8 CL#7 cells were labeled with biotin as described in Materials and Methods. Different dilutions of cell lysates involving biotinylated proteins were subjected to 10% SDS-PAGE and Western blotting followed by detection with Avidin HRP. Cell lysates were diluted as follows: Lane 1: 1/10000, Lane 2: 1/1000, Lane 3: 1/100, Lane 4: 1/10. d Immunoprecipitation of A7 antigen. Lysates of biotinylated cell-surface proteins of ROS17/2.8 CL #7 cells were subjected to immunoprecipitation using isotype control IgG or A7 MAb as described in Materials and Methods. Eluted proteins were subjected to 10% SDS-PAGE followed by transfer to PVDF membrane  and detected using Avidin HRP. No band was detected in the isotype control IgG lane. A7 MAb precipitated a cell surface antigen of molecular weight about 45 KDa. Data represent typical results among three independent experiments. e Induction time-course of A7 antigen expression in osteoblastic cells. ROS17/2.8 cells were seeded in 60 mm culture dishes. Cell lysates were prepared at 0, 1, 3, 5 and 7 days of culture. These cell lysates were subjected to quantitative western blot analysis. Time course blots were detected A7 antigen with A7 MAb (upper panel) and β-tubulin with anti-β-tubulin antibody (lower panel). Time-dependent induction of A7 antigen expression was apparent while expression level of β-tubulin did not change