MK2 regulates TNFα production posttranscriptionally. T-shFC and T-shNT cells were transfected with nontargeted siRNAs (siCon) or siRNA targeting MK2 (siMK2) by nucleofection. Cells were also subjected to nucleofection without siRNA present (mock). At the time of maximal MK2 knockdown (72 hours), the cells were stimulated with R848 (30μM) for 24 hours or were left untreated. (A) MK2 suppression (lanes 3 and 6) was confirmed by Western blot of whole-cell extracts 72 hours after nucleofection. (B) TNFα protein in supernatant media was quantified by ELISA 24 hours after R848 stimulation. siMK2 suppressed TNFα levels produced by both T-shFC and T-shNT cells and the degree of suppression matched the degree of MK2 knockdown with RNAi (6A). (C) SEAP expression, as quantified by QUANTI-Blue, was minimally suppressed by siMK2. (D) TNFα mRNA measured 24 hours after R848 stimulation by real-time qRT-PCR revealed no suppression by treatment of cells with siMK2. Therefore, MK2 suppression inhibited TNFα production and/or release, but not SEAP or TNFα gene transcription in T-shFC cells.