RIP140 deficiency impairs TLR-mediated cytokine gene expression. (A) shRNA-mediated knockdown of RIP140 expression in macrophages. Real-time PCR analysis (left) and Western blot analysis (right) of RAW264.7 macrophages infected with an adenovirus expressing a RIP140-specific or control shRNA construct. Sixty hours after infection, cells were treated with LPS (100 ng/mL) for 8 hours as indicated. Proteins were detected by Western blot using RIP140- or CREB-specific antibody. Lane 1, shRNA control; lane 2, shRNA control plus LPS; lane 3, shRNA mRIP140; lane 4, shRNA mRIP140 plus LPS. (B) Real-time PCR analysis of mRNA levels for IL-6, IL-1β, TNFα, and IFN-B1 in RAW264.7 macrophages infected with an adenovirus expressing a RIP140-specific or control shRNA construct. Sixty hours after infection, RAW264.7 cells were treated with polyI:C (10 μg/mL), LPS (100 ng/mL), or Pam3CSK4 (100 ng/mL) for 8 hours as indicated. (C) Release of IL-6, IL-1β, and TNFα from RAW264.7 cells infected and treated with LPS as in panel B. Cytokine content in the cell supernatant was determined by ELISA. Data are means plus or minus SEM (n = 9). *P < .05.