Production of sHLA-E by cytokine-activated ECs. ECs were incubated with cytokines for 48 hours or cultured for 18 hours in the absence (medium) or in the presence of IFNγ after a preincubation with CHX for 1 hours or with BrfA or galardin as a metalloproteinase inhibitor (MP inhibitor) for the last 6 hours of culture. (A) Supernatants were collected; sHLA-E was then detected by Western blotting in normal (1 ×) or concentrated (10 ×) supernatants (20 μL/sample). One representative experiment of 5 performed (B) Time-course analysis by Western blotting of sHLA-E release by ECs treated with IFNγ. Data are from 1 representative experiment of 3 performed. (C) Cells were harvested for analysis of membrane-bound HLA-E by flow cytometry following immunostaining with MEM-E/07 mAbs (solid histograms) or an isotype-matched control antibody (histograms in dotted line). MFIs are indicated above. One representative experiment of 3 performed.