FIGURE 3.

Biochemical properties of the hCLE-complex cap-binding activity. (A) Total extracts of HEK293T cells were incubated with cap analog (cap) or control resins (4B) in the presence of different concentrations of m⁷GTP, GTP or ATP (2 and 10 mM). Retained hCLE and eIF4E proteins in the corresponding resins after extensive washes were analyzed by Western blot. The experiments were repeated three times. (B) Total extracts of HEK293T cells were incubated with cap analog and control resins. After washing, protein was eluted sequentially with increasing concentrations of m⁷GTP, GTP or ATP (0.05, 0.1, and 1 mM). Eluted and resin-bound proteins were analyzed by Western blot. The experiments were repeated three times. (C) HEK293T cells were transfected with a control silencing plasmid (shCt) or with a plasmid specific for eIF4E silencing (sh4E). Total extracts were incubated with cap-analog resin alone or in the presence of GTP 2 μM and after washing, the retained proteins analyzed by SDS-gels and Western blots. (D) Total extracts of HEK293T cells were incubated with cap analog resins as indicated in part B. Protein was eluted sequentially with increasing concentrations of m7GTP, or m⁷GpppG (0.05, 0.2, and 1 mM). Eluted proteins were analyzed by Western blot and quantitated using ImageJ application (bottom). The experiments were repeated two times.