Fig 8.
RCK is involved in TTP-mediated translational repression. (A) Knockdown effects of P-body components. The effectiveness of siRNAs against Ago2 and GW182 was analyzed by Western blotting with anti-Ago2 (left) and anti-GW182 (middle). GAPDH served as a loading control. The effectiveness of Lsm1 siRNA was analyzed by qRT-PCR (right). GAPDH mRNA served as an internal control. (B) 293T cells were transfected with the indicated siRNAs and then transfected with reporter FL-GM-CSF, RL, and the indicated plasmids expressing HA-tagged TTP or HA-septin 1. Wild-type RCK resistant to siRCK (Myc-RCKr-WT) was also included in the transfections where indicated. Myc-tagged septin served as a negative control for adding back of Myc-RCKr-WT. Normalized values of FL mRNA level, activity and translation efficiency were set to 1 for cells transfected with the HA-septin 1 plasmid in each knockdown condition. Means and SD from three independent experiments are shown. **, P < 0.01. (C) Effects of RCK silencing and adding back with plasmid Myc-RCKr-WT, as determined by Western blotting with anti-RCK antibody. GAPDH served as a loading control. (D) An experiment similar to that whose results are shown in panel C was done to screen P-body components that are involved in TTP-mediated translational repression. The siRNAs used are indicated. Means and SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01. (E) An experiment similar to that whose results are shown in panel D was done to screen P-body components that are involved in TTP-mediated FL-TNF reporter mRNA translational repression. Means and SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01.