IL4I1 is a secreted glycosylated protein. (A) Whole-cell proteins (c) and medium (m) from HEK293 cells transfected with empty vector (mock) or with vectors expressing wild-type human IL4I1 (hIL4I1), mutant hIL4I1 (E481A-hIL4I1), or mouse IL4I1 (mIL4I1) were separated on a 7% NuPAGE and blotted onto PVDF. Blots were revealed with an anti-IL4I1 antibody (A left panel) or an anti-myc antibody (A right panel). (B) hIL4I1-HEK cells were treated with tunicamycin (Tuni) or equal volume of DMSO (DMSO) for the last 24 hours of culture and whole-cell proteins and medium analyzed as in panel A using an anti-IL4l1 antibody for revelation. (C) Cell lysates from mock-HEK, hIL4I-HEK, and the lymphoma cell lines MedB-1 and SU-DHL-4 (left panel) and culture media from hIL4I1-HEK and MedB-1 (right panel) were immunoprecipitated with an anti-IL4I1 antibody. Precipitates were analyzed by Western blot using the same antibody. Arrows show the position of IL4I1 and immunoglobulin heavy chain. Blots of media on right panel are 2 different exposures from the same blot.