Fig 1: Characterization of cell lines expressing different levels of HLA-DM. (A) Representative flow cytometry histograms of the cell lines used in this study. The T2, T2-DR3 cell line lacking DM, and individual clones of T2-DR3 cell lines transduced with HLA-DM were grown in standard cell culture conditions. Cells in the mid-logarithmic exponential growth phase were stained for the fluorescent proteins indicated in each case. Shaded histograms show either the non-transduced cell line (upper left, dark) or isotype controls (light gray). Stainings of the T2 cell line essentially overlap with the non-transduced cell lines (GFP) and the isotype controls (antibody stains, and therefore are not shown). (B) SDS-stable dimer assay of the cell lines shown in panel (A) by Western blot (upper panel) and quantification of the ratio stable dimer vs. DRB under conditions of no, low or high DM expression. Post nuclear cell lysates are divided and resuspended in loading buffer and either boiled (left) or kept at room temperature for 5 min (right). Samples (20 µg) are loaded and resolved in a 10% SDS-PAGE gel. The gel was subsequently blotted and probed either for detection of DRB (left, TAL 1B5 Abcam AB20181) or stably folded DR heterodimers (right, L243 produced in house), and the signal was detected in both cases using a goat-anti-mouse-HRP (Santa Cruz) and a commercial luminol-based reagent. In both cases, ß-actin detection was used as loading control using a mouse-anti-beta-actin-HRP antibody (AC-15 Abcam AB49900). The Western blot shown is a representative example of one of three independent experiments. The quantification is the result of n = 3 independent experiments measured twice each (ANOVA test: P < 0.0001).