FIGURE 3.
Inhibition of the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways by TNIIIA2. Ramos cells (3 × 106 cells) were seeded on a culture plate coated with FN and stimulated with TNIIIA2 (50 μg/ml) for different times at 37 °C. Total cell lysates were prepared and subjected to Western blot analysis using anti-phospho-ERK (A) or anti-phospho-Akt (B), as described under “Experimental Procedures.” In C, the effect of the VLA-4 antagonist, CS-1 peptide, on the phosphorylation of ERK1/2 and Akt was assessed. Cell suspension with (shown as TN) or without (shown as control) TNIIIA2 (50 μg/ml) was cultured in the presence or absence of CS-1 (200 μg/ml) for 2 h. In D, TNIIIA2-induced apoptosis was examined in Ramos cells transiently transfected with constitutively active Akt. Ramos cells were transfected with an empty vector (Cont) or cDNA for constitutively active Akt1 (CA-Akt), as described under “Experimental Procedures.” The cells were stimulated with TNIIIA2 (shown as TN or TNIIIA2) (50 μg/ml) in the presence or absence of z-VAD fmk (50 μm) and then subjected to Western blotting (panels a and b) and the cell proliferation/survival assay (panel c). *, p < 0.005 compared with control. The data are representative of three individual experiments.