Overexpression of full-length, but not truncated, Mal protein in BEL-A cells results in the expression of AnWj antigen at the cell surface. (A) Flow cytometry with mouse polyclonal anti-Mal (ab167374), human anti-AnWj eluate and monoclonal anti-AnWj (H86) wild-type BEL-A cells (top row), Mal KO BEL-A cells (second row), and BEL-A cells overexpressing either full-length Mal or truncated Mal (exons 1 and 2 only). Only BEL-A cells overexpressing full-length Mal (row 3) show any expression of Mal protein and AnWj antigen detectable by flow cytometry. (B) Full-length Mal (i), N-terminal GFP–tagged full-length Mal (ii), or GFP truncated Mal (iii; exons 1 and 2 only, as potentially expressed in the AnWj-negative patients) were expressed in KO Mal BEL-A cells and observed by confocal microscopy. Full-length Mal (ii) was located throughout the cell (but not in the nucleus) but was predominantly observed on the cell surface, whereas truncated Mal (iii) is localized inside the cell (but again not in the nucleus). Yellow scale bars are 10 μm, and white scale bars are 5 μm. Samples were imaged at 22 °C using 40× oil immersion lenses (magnification, 101.97 μm at zoom 3.8; 1.25 NA) on a TCS SP8 confocal imaging system (Leica). Images were obtained using Leica LAS AF software. KO, knockout; OE, overexpression.