RACK1 regulates the sensitivity of NF-κB activation and inflammatory reactions induced by TNF. (A) Overexpression of RACK1 postponed NF-κB target gene expression. Human 293T cells were transfected with control vectors or myc-RACK1 vectors and, 24 h later, were treated with TNF (10 ng/ml) for 0 to 60 min. The mRNA expression level of A20, IκBα, and IL8 was analyzed by RT-PCR. (B) Knockdown of RACK1 accelerated NF-κB-target gene expression. Human 293T cells were infected with control siRNA or RACK1-specific siRNA lentivirus and, 72 h later, were treated with TNF (10 ng/ml) for 0 to 60 min. The mRNA level of IκBα, A20 and IL8 was analyzed by RT-PCR. (C) Knockdown of RACK1 promoted the IκBα degradation induced by different concentrations of TNF. Control cells or RACK1-knockdown cells were treated with TNF (10 ng/ml or 0.1 ng/ml) for the indicated time. (D) Knockdown of RACK1 promoted the inflammatory gene expression induced by different concentrations of TNF. RAW264.7 cells were transfected with control or RACK1-specific siRNAs and, 48 h later, were treated with different concentrations of TNF. (E) Knockdown of RACK1 increased the TNF-induced production of TNFα and CXCL1 in primary mouse macrophages. The primary macrophages were transfected with control or RACK1-specific siRNAs, and 48 h later, were treated with different concentrations of TNF. Graphs show the mean ± SD, n = 3. *P< 0.05; **P< 0.01.