miR-29 targets TRAF4 involved in CD40-mediated NF-κB signaling. (Ai) Representative example of an immunoblot for MEC1 cells transfected with siRNA against TRAF4 (siRNA TRAF4) or negative control (siRNA NegCtrl). Seventy-two hours after transfection, the cells were stimulated with CD40L (1 µg/mL) for indicated period of time and harvested for analysis by immunoblotting. (Aii) Densitometric quantification of pIKKα/β levels for replicates of the experiment (n = 5) described in panel Ai. (Bi) Representative example of immunoblot of primary CLL cells transfected with siRNA against TRAF4 (siRNA TRAF4) or negative control (siRNA NegCtrl). Seventy-two hours after transfection, the cells were stimulated with CD40L (1 µg/mL) for indicated period of time and harvested for analysis by immunoblotting. (Bii) Densitometric quantification of pIKKα/β levels for replicates of the experiment (n = 6) described in panel Bi. (Ci) Representative example of immunoblot for MEC1 cells transfected with artificial miR-29c (miR-29 MIMIC) or negative control (miR MIMIC NegCtrl). Seventy-two hours after transfection, the cells were stimulated with CD40L (1 µg/mL) and harvested for analysis by immunoblotting. (Cii) Densitometric quantification of pIKKα/β levels for independent replicates of the experiment (n = 4) described in panel Ci. The error bars indicate the standard error of the mean. (Di) Representative example of immunoblot for primary CLL cells transfected with artificial miR-29c (miR-29 MIMIC) or negative control (miR MIMIC NegCtrl). Seventy-two hours after transfection, the cells were stimulated with CD40L (1 µg/mL) and harvested for analysis by immunobloting at the indicated time points. (Dii) Densitometric quantification of pIKKα/β levels for replicates of the experiment (n = 5) described in panel Di. (A-D) Each of the immunoblots contains 2 endogenous controls (GAPDH) marked by upper index, because for technical reasons, pIKK, TRAF4, pERK, and total ERK (+ loading control GAPDH¹) were analyzed on first gel and tIKKα and tIKKβ (+ loading control GAPDH²) on the second gel (identical protein loading and conditions). In all experiments, the statistical difference was tested using a paired t test, and the error bars indicate SEM.