Figure 4. Knockdown of LRP16 markedly attenuates NF-κB transcriptional activity.

(A) 293T cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, the endogenous expression of LRP16 mRNA was monitored by RT-PCR, and the protein levels of LRP16, p65, and p50 were determined by immunoblotting. β-actin was used as a loading control. (B–D) 293T cells were cotransfected with 3×κB-luc, siRNA and vectors as indicated. Forty-two hours after transfection, cells were stimulated with 10 ng/ml TNF-α for 7 h and then luciferase assays were performed. The relative levels of luciferase activity were normalized to the activity obtained after cotransfection of 3×κB-luc and the empty expression vector, which was arbitrarily assigned a value of 1. All experiments were performed in triplicate and were repeated at least three times, and the results are expressed as means±SD. (E) 293T cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were stimulated with 10 ng/ml TNF-α for the indicated times. Endogenous expression of the indicated genes was measured by qPCR. Data represent means±SD (error bars) of at least three independent experiments. (F) 293T cells were treated as in (E), cells were stimulated with 10 ng/ml TNF-α for 90 min. Endogenous expression of the indicated genes was measured by qPCR. Data represent means±SD (error bars) of at least three independent experiments. *P<0.05, **P <0.01, ^N P>0.05.