Western blot and immunoprecipitation analysis of transfectant-derived recombinant CD164 protein. Membrane extracts isolated from FDC-P1 cells selected with the 105.A5 and 103B2/9E10 MoAbs (105.A5 and 103B2/9E10 FDC-P1 clones) were separated by 10% SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose. The filters were successively incubated with either the 103B2/9E10 (panel 1) or 105.A5 MoAb (panel 2) supernatant (or isotype-matched, nonbinding controls), antimouse-HRPO and the immunoreactive proteins were detected by ECL as described in Materials and Methods. Under reducing conditions, MoAbs 103B2/9E10 and 105.A5 identify similar recombinant proteins of 80 kD. (A1) and (A2) contain reduced 103B2/9E10 and 105.A5 FDC-P1 protein blotted, respectively. Lanes (a) IgG₃ negative control, (b) MoAb 103B2/9E10, (c) IgM negative control, and (d) MoAb 105.A5. (B) Biotinylated membrane preparations of FDC-P1 cells expressing CD164 (ie, 103B2/9E10 and 105.A5-selected FDC-P1 cells) were coincubated with the indicated MoAb for 16 hours at 4°C. Immune complexes were precipitated using goat antimouse sepharose, resuspended in reducing SDS-PAGE buffer, resolved on a 7.5% SDS-polyacrylamide gel, and visualized with biotin-streptavidin-HRPO complex and ECL. MoAb 103B2/9E10 immunoprecipitate a protein of the appropriate molecular mass from lysates derived from both 105.A5 and 103B2/9E10 FDC-P1 cells. Lane 1, 103B2/9E10 FDC-P1 lysate immunoprecipitated with MoAb 103B2/9E10; lane 2, 105.A5 FDC-P1 lysate immunoprecipitated with MoAb 103B2/9E10; lane 3, 103B2/9E10 FDC-P1 lysate immunoprecipitated with IgG₃ negative control; lane 4, 105.A5 FDC-P1 lysate immunoprecipitated with IgM negative control.