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. 2009 Mar 9;29(10):2505–2520. doi: 10.1128/MCB.00034-09

FIG. 6.

FIG. 6.

PRL-induced tyrosine phosphorylation of Grb2 inhibits EGF-induced Ras/MAPK activation. (A, left) Serum-starved differentiated HC11 cells were pretreated with sodium vanadate for 30 min and were left unstimulated or stimulated with EGF (E), PRL (P), or PRL/EGF (PE) for 15 min. Cell lysates were immunoprecipitated using a polyclonal antibody to Grb2, immunodetected with a monoclonal antibody to phosphotyrosine, and reprobed with a polyclonal antibody to Grb2. (Right) Differentiated HC11 cells were transfected with an expression vector encoding myc-Grb2. Serum-starved cells were pretreated with sodium vanadate and stimulated as described above. Cell lysates were immunoprecipitated using a polyclonal antibody to phosphotyrosine and immunodetected using a monoclonal antibody to the myc epitope (upper panel). Cell lysates were immunoblotted with a monoclonal antibody to the myc epitope (lower panel). (B) Differentiated HC11 cells were transfected with a vector or a plasmid encoding myc-Grb2YF. Starved cells were stimulated with EGF or PRL/EGF as indicated. Numbers indicate time (in minutes). Lysates were immunoblotted using a polyclonal antibody to phospho-Erk1/Erk2 (upper panel) and Erk1/Erk2 (lower panel). (C) HC11 cells treated as described above were stimulated with EGF or PRL/EGF as indicated. A pulldown assay of Ras-GTP was performed, and immunodetection was done using a monoclonal antibody to Ras (upper panel) and a polyclonal antibody to GST (middle panel). Cell lysates were immunodetected with a monoclonal antibody to Ras (lower panel). (D) Differentiated HC11 cells were transfected as described above. Serum-starved cells were left unstimulated or were stimulated with EGF or PRL/EGF for 15 min. Lysates were immunoprecipitated with a polyclonal antibody to Grb2 or to myc, immunoblotted with a monoclonal antibody to Sos (upper panel), and reblotted with a polyclonal antibody to Grb2, or total cell lysates were immunodetected using a monoclonal antibody to myc (lower panels). α-, anti-; PY, phosphotyrosine; TCL, total cell lysates.