IFN-β influences the interaction of PTP1D/Syp and other tyrosine-phosphorylated proteins with GST-Grb2 fusion proteins. (A) Grb-2 was immunoprecipitated from lysates prepared from U266 cells (8 × 10⁶) treated with IL-6 and/or IFN-β as indicated. Associated proteins were analyzed by immunoblotting with anti-pTyr and anti-PTP1D/Syp specific MoAbs. Equivalent loading of Grb2 was confirmed by final reprobing of this blot with a Grb2-specific MoAb (bottom panel). (B) Extracts from U266 cells (1 × 10⁷) treated with IFN-β and IL-6 as indicated were incubated with a GST-Grb2 fusion protein (approximately 10 μg) bound to glutathione-Sepharose beads for 2 hours at 4°C. Beads were washed three times with lysis buffer, associated proteins eluted in SDS-sample buffer, and analyzed by immunoblotting with anti-pTyr and anti-PTP1D/Syp MoAbs. (C) The experiment in (B) was repeated using a GST-Grb2SH2 fusion protein (GSTSH2). (D) The experiment in (B) was repeated using control GST protein. Arrows indicate the position of PTP1D/Syp and other tyrosine-phosphorylated proteins discussed in the text.