Fig 6. US2-associated proteins identified by modulating CypC expression participate in US2 degradative functions.

(A) Determination of siRNA knockdown efficiency. The indicated targets of interest were depleted in U373-MG + US2 cells using 2–3 individual siRNAs transfected together on day 0 and boosted on day 3. On day 6 cells were lysed and RNA isolated for real-time PCR analysis. All knockdowns resulted in 90% or greater depletion of target mRNAs. Results were normalized to beta actin using the delta delta Ct method for comparison of knockdown treatments. An unpaired Student’s T-test was used to show statistically significant differences from the negative control siRNA (* indicates p-value < 0.05, n = 5). (B) Using a similar method, HLA-A transcripts were quantified under various knockdown conditions. (C) U373-MG + US2 cells and U373-MG control cells were treated with the indicated siRNA pools and on day 6 MHC class I levels were determined by immunoblotting with mAb HC10. (D) Quantified MHC I band intensities from the experiment in panel C. All results were normalized to the negative control siRNA sample for each cell line (* indicates p-value < 0.05, n = 4). (E) U373-MG + US2 cells and U373-MG control cells were subjected to knockdown as in panel (C) and surface MHC I levels were determined by flow cytometry following staining with mAb W6/32.