Intracellular localization and cleavage analysis of XXYLT1. Images A–E show immunostainings of CHO cells that were transiently transfected with tagged plasmids. A, co-localization of the single-tagged constructs FLAG-XXYLT1 and XXYLT1-HA. B, overlaying signals from the N-terminal FLAG tag and the C-terminal HA tag of the FLAG-XXYLT1-HA construct. C, co-localization of XXYLT1 visualized by the FLAG tag and a co-transfected GFP1-KDEL as ER marker. D, no merge was observed after staining of tagged XXYLT1 and the Golgi marker mannosidase II. E, as control, the established Golgi-located β1,4-galactosyltransferase (FLAG-B4GALT1-HA) was investigated in parallel. F, signal sequence prediction (SignalP 3.0) indicated a high probability for XXYLT1 to have a cleaved signal sequence, which is not predicted anymore in the FLAG-tagged construct. G, lysates of cells transiently expressing FLAG-XXYLT1-HA, the control protein FLAG-B4GALT1-HA, as well as C-terminally HA-tagged enzymes were analyzed by Western blotting using either anti-HA or anti-FLAG antibody, showing no indication of cleavage of the protein. XXYLT1 partly runs as an SDS-resistant dimer.