Functional UFM1 is required for efficient HLA-I dislocation to the cytosol. (A) U373 cells stably expressing HLA-A2-eGFP, US2, and Cas9 were transduced either with sgUFM1 or with an empty vector control (sgCtrl). HLA-A2-eGFP expression of the polyclonal cell population was assessed by flow cytometry at 14 days post-transduction. (B) The U373 cells shown in A were treated overnight with the proteasome inhibitor MG132 or DMSO control, after which they were lysed for western blot analysis. The cell lysates were treated with EndoH, PNGaseF, or control as indicated. This experiment was performed three times, of which one representative blot is shown. (C) Quantification of (B). The change in proportions of glycosylated/non-glycosylated HLA-I upon introduction of sgUFM1 (lane 9 versus 12) is significant (Fisher’s exact test, * p < 0.05).