Validation of TRAF4 as a target of miR-29. (Ai) Representative example of immunoblot for TRAF4 levels in MEC1 cells transfected (72 hours) with siRNA against TRAF4 (siRNA TRAF4), control siRNA (siRNA NegCtrl), artificial miR-29c (miR-29 MIMIC), or control miRNA (miR MIMIC NegCtrl). (Aii) Densitometric quantification of TRAF4 levels for independent replicates of the experiment (n = 5) described in panel Ai. (B) Analysis of TRAF4 mRNA levels in MEC1 cells transfected by synthetic miR-29 or siRNA against TRAF4 (n = 5) as described in panel A. (Ci) Representative example of immunoblot analysis for TRAF4 levels in primary CLL cells transfected (72 hours) with siRNA against TRAF4 (siRNA TRAF4), control siRNA (siRNA NegCtrl), artificial miR-29c (miR-29 MIMIC), or control miRNA (miR MIMIC NegCtrl). (Cii) Densitometric quantification of TRAF4 protein levels for independent replicates of the experiment (n = 4) described in panel Ci. (D) Analysis of TRAF4 mRNA expression in primary CLL cells transfected by synthetic miR-29 or siRNA against TRAF4 (n = 4) as described in panel Ci. (E) Alignment of miR-29a/b/c with 3′UTR of TRAF4 mRNA (at position 161-167). (F) Luciferase activity in HEK293FT cells cotransfected with psiCHECK2 vector containing the cloned 3′UTR region of TRAF4 encoding the putative miR-29 binding site at position 161-167 (TRAF4 UTRwt) and either artificial miR-29c (miR-29 MIMIC) or control miRNA (mimic NC), or containing a cloned mutated 3′UTR of TRAF4 (TRAF4 UTRmut; (G>C at position 165) and either artificial miR-29c (miR-29 MIMIC) or control miRNA (mimic NC). Renilla activity was measured 24 hours after transfection, and activity was normalized to the endogenous firefly control of the psiCHECK2 vector (n > 5). In all experiments, the differences were compared by paired t test. The error bars indicate SEM.