FIGURE 6.

Biochemical analysis of DO proteins produced by the DOα variant alleles.

(A) HeLa.CIITA cells were transiently transfected with vectors encoding DOB*0101 and DOA*0101 or one of the DOα variants with altered function (DOA*0102, DOA*0103, DOA*0104, I67M, V92M, R97W, P103T, F114L, R124H, G126R, R147C, and H150R; labeled across the top of the blots). Mock transfected HeLa.CIITA cells (Mock) were used as a negative control. Cells were harvested 72 hours later, lysed, and DO was immunoprecipitated with the DO heterodimer specific antibody Mags.DO5 (33). The resulting precipitated proteins (α-DO-IP) were separated by SDS-PAGE, transferred to membranes and western blotted to determine DOβ (R.DOB/c (34) or DMα protein (clone EPR7981) levels. Western blot analyses of the lysates (Total) used for the immunoprecipitations are included to confirm protein expression (bottom).

(B and C) Quantification of the amount of DOβ (B) and co-associated DMβ (C) protein detected in each α-DO immunoprecipitation. Values were either normalized to correct for transfection efficiency based on the number of mRuby+ AcGFP+ cells in each sample. Values were then normalized to the amount of DOβ or DMβ protein obtained after transfection of DOA*0101/DOB*0101 (black bar) to allow for comparison across four independent experiments.

(D) Ratio of the amount DOβ to DMβ obtained for each α-DO immunoprecipitation combined for the 4 independent experiments. Values from (B and C) were normalized to the ratio obtained after transfection of DOA*0101/DOB*0101 (black bar) to allow for comparison across the four independent experiments.

(E-G) HeLa.CIITA cells were transiently transfected with vectors encoding DOB*0101 and DOA*0101, one of a few select DOα variants with altered function (DOA*0102, DOA*0103, DOA*0104, and H150R) or as negative controls EV-mRuby only or no vectors (mock). Cells were harvested 72 hours later, and mRuby+ AcGFP+ cells were purified by cell sorting, except for the mock transfection. (E) Western blotting of lysates verified DO and DM expression in the cells. (F) Equal numbers of cells were lysed, and the resulting lysates were split into two equal aliquots. DO was depleted by three sequential anti-DO immunoprecipitations with Mags.DO5 (33) and from one aliquot and as a negative control, three sequential immunoprecipitations were performed using mouse IgG. The resulting precipitated proteins were separated by SDS-PAGE, transferred to membranes and blotted to confirm DO depletion by blotting for DOβ (R.DOB/c (34)). (G) Control (C) and DO-depleted (IP) supernatants from the third immunoprecipitation were probed for DM (DMβ; clone EPR7981) to determine if any DM remained after DO removal.