Selective response of CLEC-2–expressing 293T-REx cells to rhodocytin. (A) 293T-REx cells, which express CLEC-2 under a tet repressor, were incubated with vehicle or 1 μg/mL of doxycycline for 24 to 48 hours. 5 × 106/mL cells were incubated with an antibody to CLEC-2 or an isotype matched control and analyzed by FACscan. Filled areas represent vehicle-treated 293TRex cells; lines, doxycycline-treated 293TRex cells. (B) 293T-REx cells (5 × 106/mL) pretreated with or without doxycycline were preincubated with 100 nM rhodocytin. After excess of rhodocytin was removed by centrifugation, cells were incubated with control rabbit IgG (filled areas) or antirhodocytin antibody (lines), followed by FITC-conjugated anti–rabbit IgG. (C) 1 × 107 cells were dissolved with 4 × SDS sample buffer. Proteins were separated by SDS-PAGE and blotted with anti–CLEC-2 antibody (left panel) or control–goat IgG (right panel). Lane 1: vehicle-treated 293TRex cells; lane 2: doxycycline-treated 293TRex cells. (D) Cells were stimulated with or without 500 nM rhodocytin for 10 minutes, dissolved with SDS sample buffer, and separated by SDS-PAGE. Protein tyrosine phosphorylation was detected by Western blotting with antiphosphotyrosine antibody (4G10). The data are representative of 2 to 3 experiments.
