FIGURE 1.

Approach used to measure DO function.

(A) HeLa.CIITA cells (MHCII⁺, Ii⁺ and DM⁺) were transiently transfected with an empty mRuby vector (EV-mRuby) and empty AcGFP vector (EV-AcGFP), a vector expressing DOA*0101 mRuby and EV-AcGFP, EV-mRuby and a vector expressing DOB*0101-AcGFP, or DOA*0101-mRuby and DOB*0101-AcGFP. Cells were analyzed 72 hours later by flow cytometry after staining for cell surface MHCII and MHCII-CLIP expression. Live cells were gated for the expression of both mRuby which reports DOA expression and AcGFP which reports DOB expression as shown. MHCII levels in the mRuby+ AcGFP+ cells are shown (left) and after gating for the MHCII⁺ cells, MHCII-CLIP levels are shown (right). Geometric mean fluorescent intensity (gMFI) values for MHCII and MHCII-CLIP are shown on each histogram.

(B) HeLa.CIITA cells transfected as in (A) were fixed, permeabilized and stained for intracellular DM and DO prior to analysis by flow cytometry. Fixed cells were gated on AcGFP⁺ mRuby⁺ cells (fixation of cells results in decreased mRuby and AcGFP fluorescence) and the DM levels determined (left). After gating for DM⁺ cells, the level of DO was determined (right). gMFI values for DM and DO are shown on each histogram. DO expression and increased MHCII-CLIP levels were observed only after transfection of vectors expressing both DOA and DOB. Data is representative of five similar experiments.