Figure 3. Characterization of NIH 3T3 variants with various levels of Ras-GTP.

Dominant-negative (S17N) or constitutively active (G12V) H-Ras was introduced in NIH 3T3 cells by retroviral infection and selection, with empty vector serving as a control. (A) H-Ras expression. Cell lysates (25 μg of total protein) were subjected to SDS/PAGE and blotted for H-Ras. (B) Ras-GTP levels. Cell variants were unstimulated (grey bars) or stimulated with PDGF (black bars). Ras-GTP levels were measured as in Figure 2(B) and were normalized by the level in unstimulated control cells (approx. 18000 molecules/cell, determined from GTP standards). Values are means±S.E.M.; n=2. (C) PDGF receptor phosphorylation. Cells were stimulated with the indicated concentrations of PDGF (nM) for 5 min, and lysates were probed with anti-phospho-PDGFβ-receptor (pTyr⁷⁵¹) antibodies. (D) ERK phosphorylation. Lysates were prepared from cells stimulated with the indicated concentrations of PDGF for 5 min, subjected to SDS/PAGE and probed with anti-phospho-ERK (pThr²⁰²/pTyr²⁰⁴) antibodies. The blot shown is representative of three independent experiments. (E) MKP-1 expression. Lysates were prepared from cells stimulated with the indicated concentrations of PDGF (nM) for 5 min, subjected to SDS/PAGE and probed with anti-MKP-1 antibodies.