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. 2013 Jul 3;34(12):2880–2890. doi: 10.1093/carcin/bgt238

Fig. 6.

Fig. 6.

Targeting of the phosphorylated Src*/SD to membranes promotes a transformed phenotype in vitro. (A) Schematic of the Src*/SD constructs. AA, amino acids. CAAX, H-Ras lipid anchor. P, phosphorylation. (B–D) 3Y1 rat fibroblasts expressing the Dox-inducible constructs for EV, Src*/SD (SD-WT) and Src*/SD-CAAX (SD-CAAX) were analyzed after 2 days (B and D) and 5 days (C) of Dox induction (2 μg/ml). (B) Phosphorylated SD-CAAX is targeted to the membrane fraction in 3Y1 cells. WCE were fractionated into cytoplasmic (S) and membrane/pellet (P) fractions and analyzed by WB with CasB, β-actin and pTyr antibodies. Lower panel, brackets indicate proteins differentially tyrosine phosphorylated in EV, SD-CAAX and SD-WT cells. (C) Equal cell numbers (10 000) expressing the indicated constructs were seeded in 6-well plates and focus formation was analyzed by phase contrast microscopy after 5 days. Scale bars, 250 μm. (D) Upper panel, Erk1/2 phosphorylation was analyzed by IB in WCE (40 μg) of 3Y1 cells expressing the indicated constructs. Total ERK1/2 and β-actin antibodies were used as controls. Lower panel, the fold change in pErk1/2 was determined by densitometric analysis of two independent experiments setup in duplicates. n.s., not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.