FIG. 2.

Ephrin-B1 stimulation of COS-1 cells leads to tyrosine phosphorylation of endogenous EphB2 and down regulation of ERK1/2 phosphorylation. (A) COS-1 cells were serum starved for 14 h and were either left untreated (lane C) or stimulated with 8 μg of clustered human Fc (lanes Fc) or Fc-ephrin-B1 (lanes Ephrin-B1) per ml. The cells were subsequently washed twice with PBS and lysed in PLC buffer as indicated in Materials and Methods. Lysates equalized for protein concentration were subjected to immunoprecipitation (IP) with anti-EphB2 antibodies, and immunoprecipitates were subsequently separated by SDS-PAGE and blotted with anti-pTyr antibodies (upper panel). The membranes were then stripped and reprobed with anti-EphB2 antibodies (bottom panel). (B) Serum-starved (left panels) and growing (right panels) COS-1 cells were challenged for the indicated time with 8 μg/ml clustered Fc or Fc-ephrin-B1 and subsequently lysed and immunoblotted (WB) with anti-phospho-ERK1/2 antibodies as indicated in Fig. 1. The membranes were then stripped and reprobed with anti-ERK1 antibodies (B, bottom panels).