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

FIG. 7.

FIG. 7.

Tyrosine phosphorylation of Grb2 is essential for the inhibitory effects of PRL on EGF-induced cell proliferation. (A) HC11 cells were transfected with a vector (V) or a plasmid encoding myc-Grb2YF (YF). Cell lysates were immunoblotted with a monoclonal antibody to the myc epitope to detect stable expression of myc-Grb2YF. (B) Serum-starved differentiated HC11-vector1 (left) or HC11-Grb2YF2 (right) clones were treated with PRL (P), EGF (E), or PRL/EGF (PE), as indicated. Numbers indicate time (in minutes). Cell lysates were immunoblotted with a polyclonal antibody to phospho-Erk1/Erk2 (upper panels) and Erk1/Erk2 (lower panels). Blots shown are representative of HC11-vector and HC11-Grb2YF isolated clones. (C) Vector or Grb2YF-transfected HC11 cell lines were grown o/n in media containing 2% serum HI or HIP. Cells were then left untreated or were treated with EGF for 72 h. An MTT assay was performed, and the results are expressed as the means ± SEM for triplicates of three different experiments (*, P = 0.048). (D) Vector or Grb2YF HC11 clones were grown o/n in media containing 2% FBS HI or HIP. Cells were then left untreated or were treated with EGF for 24 h and 48 h. A cell counting assay was performed, and the results are expressed as the means ± SEM for triplicates of three experiments (*, P = 0.034; **, P = 0.041). (E, left) 293 cells were transfected with either myc-tagged Grb2 or Sil-Grb2YF in the absence or the presence of pU6+27-ShGrb2. At 48 h following transfections, cells were lysed and immunoblotted with a monoclonal antibody to myc or a monoclonal antibody to β-tubulin. (Right) HC11 cells were cotransfected with a vector, a vector and pU6+27-ShGrb2, or the Sil-Grb2YF mutant and pU6+27-ShGrb2. Transfected HC11 cells were selected in media containing G418 for 48 h. Cells were then grown o/n in media containing 2% FBS HI or HIP. Cells were then left untreated or were treated with EGF for 48 h. An MTT assay was performed, and the results are expressed as the means ± SEM for triplicates of three experiments (*, P = 0.002; **, P = 0.0017). Abs, absorbance; α-, anti-; TCL, total cell lysates.