Fig. 5. Effects of SHPS-1 ligands on tyrosine phosphorylation of SHPS-1 (A–C), on the association of SHPS-1 with SHP-2 (A), and on tyrosine phosphorylation of FAK (D) and paxillin (E). WM239a cells were incubated at 37°C for 30 min (A, D, E) or for indicated times (B) with control mouse IgG (5 µg/ml) or with the SE12C3 mAb to human SHPS-1 (5 µg/ml), as indicated. Alternatively, the cells were incubated with control human IgG (10 µg/ml) or human CD47-Fc (10 µg/ml) for 30 min at 37°C (C). Whole-cell lysates were then prepared and subjected to immunoprecipitation (IP) with the SE12B6 mAb to SHPS-1 (A, B, C), a mAb to FAK (D), or a mAb to paxillin (E). The resulting precipitates were subsequently subjected to immunoblot analysis with either horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine (αpY), a mAb to SHP-2, pAbs to SHPS-1, pAbs to FAK, or a mAb to paxillin, as indicated. Results are representative of three independent experiments.