iPsiRNA-mediated down-modulation of inducible iP subunits in DCs. (A) iDCs were electroporated with the indicated siRNAs (500 nM of each individual iP subunit–specific siRNA, with control siRNA added as needed to a final total concentration of 1500 nM). At 4 hours later, LPS was added to the siRNA-transfected DCs. After 24 hours of maturation induction, DC RNA was extracted and quantitative PCR was performed to determine relative levels of mRNA encoding iP subunits LMP2, LMP7, and MECL-1. The data presented are representative of 3 separate experiments, each with similar results. (B) Using DCs derived from CD14⁺ monocytes, intracellular expression of cP subunits X and Y and iP subunits LMP2 and LMP7 was assessed using specific mAbs, as well as isotype-matched control mAbs, and an APC-conjugated secondary antibody, detected as FL4. In the top panel, iDCs and mDCs (induced to mature using a CC for 24 hours in culture) were analyzed, while in the bottom panel, the DCs analyzed were transfected with either CsiRNA or iPsiRNA and induced to mature for 24 hours using the CC. The data presented are representative of 2 experiments, each with similar results. (C) Specific subunit composition of 20S proteasomes from mDCs transfected with either CsiRNA or iPsiRNA was assessed by Western blotting of cell lysates. Densitometry measurements were compared as indicted. This experiment was performed 3 times, with similar results.