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. 2006 Jun 8;25(12):2781–2791. doi: 10.1038/sj.emboj.7601166

Figure 5.

Figure 5

RNAi-mediated silencing of RSK1 and RSK2 isoforms expression leads to reduced eIF4B Ser422 phosphorylation and inhibition of cap-dependent translation. (A) HeLa cells were subjected to RNAi using synthetic oligos nonspecific (Mock) or specific to RSK1 and RSK2 isoforms. At 24 h post-transfection, cells were serum starved for 16–18 h in the presence or absence of rapamycin, then indicated samples were treated with U0126 and stimulated with serum or insulin as shown. Total cell extracts were immunoblotted with phospho-eIF4B S422 and phospho-ERK1/2 T202/Y204 antibodies followed by reprobing for the corresponding total proteins. RSK1 and RSK2 Western blots were also carried out to demonstrate the efficiency of the knockdown. (B) HEK293 cells were transfected with the bicistronic luciferase construct and indicated siRNAs. After 48 h, cells were harvested and assayed for Renilla (RL) and firefly (FL) luminescence. Results are presented as average of RL/FL ratio±standard error from three independent experiments carried out in triplicate.