SP-A enhances BCG-induced ERK1/2 MAP kinase activation. Panel A: RBMM were incubated with BCG or SP-A-BCG complexes as described in Figure 1 for 0–30 min. At each time point, cells were washed with cold PBS containing 100 μM sodium vanadate to remove any uningested BCG and to inactivate phosphatase activity. Cells were solubilized in immunoprecipitation buffer and total proteins were isolated. Extracts were analyzed by SDS-PAGE, followed by transfer to nitrocellulose, and analysis by Western blot using an antibody specific for phosphorylated forms or ERK-1 and ERK-2. Panel B: RBMM were incubated for the indicated times with BCG, SP-A-BCG, or SP-A alone. Total protein was extracted as described above. Activated ERK-1/2 was immunoprecipitated using a phospho-specific antibody. The antibody-ERK-1/2 complex was then added to a mixture containing ATP and a GST-Elk-1 fusion protein and allowed to incubate for 5 min. The proteins were separated by SDS-PAGE and phosphorylated Elk-1 was visualized by Western blot analysis. Panel C: bands from the blots shown in panel B corresponding to phosphorylated Elk-1 after 5 min treatment with immunoprecipitated ERK-1/2 were quantified using image analysis. Blots are representative of three independent experiments and were normalized for equal protein loading by Western blot analysis for non-phosphorylated proteins within the same membrane.