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. 2015 Jan 9;6(6):4080–4096. doi: 10.18632/oncotarget.2977

Figure 4. TRAF4 promotes the phosphorylation of p70s6k through enhancing the interaction between mTOR and p70s6k.

Figure 4

(A) TRAF4 promotes the phosphorylation of p70s6k and S6. The Western blot analysis indicated that overexpression of TRAF4 in MCF7 significantly increased the phosphorylation of p70s6k/S6 when compared with control vector. While, knocking down of TRAF4 in MDA-MB-231 significantly reduced phosphorylation level of p70s6k/S6 when compared with negative control. (B) TRAF4 promotes the phosphorylation of S6 through p70s6k. p-S6 expression significantly increased after TRAF4 plasmid was transfected into MCF7. while, elevation of p-S6 expression was obviously inhibited when TRAF4 expression vector together with p70s6k specific siRNA were transfected into MCF7. (C) Zinc fingers domain of TRAF4 is responsible for its promoting effect on p70s6k/S6 signaling. The protein levels of p-p70s6k and p-S6 were significantly increased after the expression vectors for wild type TRAF4(WT), ΔR, and ΔT mutants were transfected into MCF7 cells when compared with control empty vector. But, there were no obvious changes on p-p70s6k and p-S6 protein level after Flag-tagged-ΔZn mutant was transfected into MCF7 when compared with control empty vector. (D) mTOR activity is predominantly required for the promoting effect of TRAF4 on enhancing activation of p70s6k/S6 pathway. The elevation of p-p70s6k and p-S6 expression was obviously inhibited when TRAF4 overexpression together with Rapamycin treatment. The relative expression levels of protein were calculated in reference to Actin as the loading control. (E) TRAF4 promotes the interaction of p70s6k and mTOR. The relative expression of p70s6k binding to mTOR was calculated in reference to mTOR as the loading control. (F) Rapamycin inhibits the interaction between TRAF4 and p70s6k. The expression of TRAF4 that binding to p70s6k was siginificantly reduced in Rapamycin group than that in control group. The relative expression of TRAF4 binding to p70s6k was calculated as (E) Data was presented as mean ± SD from three independent experiments. (*p < 0.05).