Fig. 6.

Functionality of selected DARPins in living cells. (Upper) BRET² assays were performed in COS-7 cells 24 h after transfection of the indicated fusion constructs at a 1:1 DNA ratio. Before measurements, cells were starved for 6 h in serum-free media and treated for 10 min with PBS (−), 100% FBS, or 10 μM of the MEK1 inhibitor PD98509. The background BRET² signal deriving from control transfections with vector coding for unfused Rluc is indicated by dashed lines. Data are means with SEM of at least three independent experiment with n = 6. ***P < 0.001. (Lower) Levels of phospho-Rluc-ERK2 (Rluc-pERK2) and Rluc-ERK2 were monitored for the different treatments by Western blot, using anti–phospho-p44/42 (α-pERK) and anti-Rluc (α-Rluc) primary antibodies.