(A) MDCK-RAF1:ER cells were subjected to 6 μM fmk pulse (1 h fmk incubation followed by extensive washing), 10 μM BI-D1870, 100 μM SL0101, 5 μM GF109203X and 1 μM 4HT as indicated, and photographed after 24 h.
(B) Lysates or medium from cells treated as in (A) were analysed by immunoblotting.
(C) MDCK-RAF1:ER cells transfected with plasmid expressing HA-tagged RSK1, -2, -3 or -4 were exposed to 6 μM fmk and 1 μM 4HT, as indicated. After 24 h, the cells were analyzed by immunoblotting.
(D) MDCK-RAF1:ER cells stably expressing wild-type RSK2 or fmk-resistant RSK2-C436V mutant were analyzed in multilayering assays or by immunoblotting.
Experiments were conducted 3 times with similar results.