Figure 6.

Perturbation of the MAPK signaling pathway by PA-mediated delivery of an affibody (Lv6) that targets Raf. A) HEK293T cells were transfected with pcDNA3-ABRaf for 24 h, starved overnight, treated with 5 ng mL⁻¹ EGF for 7 min, lysed with buffer containing 1 % NP-40, and subjected to anti-pErk1/2 immunoblotting. The membrane was stripped and re-blotted with anti-Erk1/2 antibody to serve as loading control. B) Cytosolic fractions extracted by digitonin from HEK293T cells treated with 500 nm Lv6 and 80 nm PA or PA[F427H] in serum-free medium for 12 h (∼400 000 cells per lane). C) HEK293T cells treated with 500 nm Lv6 and 80 nm PA or PA[F427H] in serum-free medium for 12 h, then with 5 ng mL⁻¹ EGF for 7 min, lysed, and analyzed as in (A). Bar graph: quantification of phosphorylation of Erk1/2 (pErk1/2; n=3); bars correspond to the lanes above. pErk1/2 bands normalized to that of Erk1/2, and compared to cells treated with EGF (set to 1). Data are averages of three experiments. P values: Student's t-Test.