Cellular Expression of A20 and ABIN-3 in Response to Toll-Like Receptor-4 Stimulation

Although Toll-like receptor (TLR)-induced expression of several proinflammatory genes is required to provoke an efficient immune response, excessive or prolonged activation of TLR signaling can contribute to the development of septic shock and several inflammatory diseases. Given this inherent danger of unrestrained TLR signaling to the organism, it is not surprising that many negative feedback mechanisms have evolved to hold TLR signaling in check. In this context, TLR stimulation induces several negative regulators of TLR-induced signaling to nuclear factor (NF)-κB dependent gene expression. Here we describe the use of Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) to study respectively the cellular protein and mRNA expression levels of the NF-κB inhibitory proteins A20 and ABIN-3 in response to TLR4 stimulation by lipopolysaccharide (LPS).