The SH2 domain of Lnk is required to inhibit Epo-dependent growth and signaling pathways in 32D/EpoR cells. (A) Overexpression of Lnk in 32D/EpoR cells inhibits cell growth in response to Epo. We introduced either vector alone or wild-type Lnk into 32D/EpoR cells and determined the proportion of infected cells as those that express GFP, 2 days later. We then measured the GFP⁺ percentages and counted total cell numbers every day. The numbers of vectoror Lnk-expressing GFP⁺ cells were then calculated and plotted (mean ± SD). (B) We introduced the wild-type or the mutant Lnk cDNA constructs into 32D/EpoR cells. Two days after infection, we cultured the cells in 1 U/mL Epo, and measured the GFP⁺ fraction every 3 days thereafter. The percentage of GFP⁺ cells relative to that at 2 days after infection was plotted. Results shown are representative of more than 5 independent experiments. (C-D) 32D/EpoR cells infected with either vector alone or the wild-type or the mutant forms of Lnk were purified, stimulated with Epo, and lysed at indicated intervals followed by Western blotting analysis. (C) Stat5 phosphorylation and protein levels after Epo administration. (D) P42/44 MAPK phosphorylation and protein levels after Epo administration. Representatives of 3 independent experiments are shown.