Signal transduction in OPN-treated CD34⁺ cells. (A) Purified CD34⁺ cells were incubated with 1 μg/mL OPN in the presence or absence of a neutralizing antibody against OPN for 2 minutes. The cells were fixed and permeabilized, followed by incubation with FITC-conjugated antiphosphotyrosine antibody (4G10) and analysis by flow cytometry. Cells were gated for forward and side scatter as shown in the inserted panel. Solid and dashed lines in the histogram show the degree of phosphorylation of cellular proteins in the absence and presence of the neutralizing antibody, respectively. Black filled area shows untreated control cells. (B) Immunoblot analysis of tyrosine-phosphorylated proteins in CD34⁺ cells. Total protein extracts of untreated or OPN-treated CD34⁺ cells were analyzed for tyrosine phosphorylation by Western blot. CD34⁺ cells were cultured for 2 minutes in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 μg/mL OPN. Total protein extracts were prepared, run on SDS-PAGE under reducing conditions, and transferred onto a nitrocellulose membrane. The membrane was stained with Ponceau S (Sigma, St. Louis, MO) to show protein load and incubated with PY20 antiphosphotyrosine antibody. An arrow indicates the position of the tyrosine-phosphorylated protein (34 kDa) that has increased phosphorylation after exposure to OPN. (C) CD34⁺ cells were cultured for 2 minutes in the absence (none) or presence of 1 μg/mL OPN (+OPN), OPN plus 13.5 μg/mL of a neutralizing antibody (+OPN+Ab), or OPN plus 13.5 μg/mL mouse IgG1 (+OPN+isotype). Total protein extracts were prepared, and tyrosinephosphorylated proteins were analyzed by immunoblots. The band intensity of the tyrosine-phosphorylated 34-kDa protein was measured by phosphor image analysis. Comparable results were obtained from 3 different cell preparations.