Figure 2.

Time course of GPI-induced tyrosylphosphorylation and identification of the PTK. (A) LPS-nonresponsive macrophages were treated with GPI for the indicated times and phosphotyrosyl proteins detected by Western blot analysis. (B) In vitro kinase assay. Cells were incubated with 10 μM GPI and the reaction was stopped at various times. Lysates were precleared with rabbit preimmune serum, followed by immunoprecipitation with specific antisera to p59^hck, and the immune complexes were subjected to in vitro kinase assay with exogenous ATP-γ ³³P and polyGlu/Tyr as substrate, followed by SDS/PAGE and fluorography. (C) Independent experiment conducted as in B but using 1 μM GPI, cold ATP, acid-denatured enolase as exogenous substrate, and detection of phosphotyrosine or hck by Western blot analysis (WB).