Fig. 4.

Affinity precipitation. ERK and pERK were immunoprecipitated from human embryo kidney (HEK293T) cells by the selected ERK, pERK, and ERK/pERK DARPins indicated. Precipitated ERK2 and pERK were detected by Western blot, using an ERK2- and pERK-specific antibody. Tested DARPins precipitate unphosphorylated, phosphorylated, or both ERK forms from cell lysates, giving rise to the corresponding bands on a Western blot, whereas the controls, the nonbinding DARPin E3_5 and beads with no DARPin, did not. The presence of DARPins was confirmed by detection of the N-terminal RGS-His tag. Note that E39, E40, and E3_5 are N3C molecules, which because of their stability do not completely denature in SDS/PAGE.