Comparative characterization of Xq22.3 Env, Syncytin-1, MSRV Env, and reconstituted full-length Xq22.3 Env. (A) Protein lysates from HeLa cells transfected with the indicated HERV-W Env vectors were treated (+) or not treated (-) with peptide-N-glycosidase (PNGase F) to investigate glycosylation of the different HERV-W Env proteins. (B) Protein lysates were generated under reducing (+) or non-reducing (-) conditions to study oligomerization of HERV-W Env proteins. Immunoblots were incubated with the indicated primary antibodies. (C) HeLa cells were grown on microscope slides and transfected with MSRV Env, Xq22.3 Env FLΔStop, and Xq22.3 Env. Surface (SF) expression of the respective proteins was investigated by immunocytochemistry of living, unfixed, and unpermeabilized cells. Intracellular (IC) expression was analyzed in fixed and permeabilized cells. Monoclonal antibody 13H5A5 was used as primary antibody. Magnification × 1000. (D) Flow cytometry was performed on HeLa cells transfected with MSRV Env, Xq22.3 Env FLΔStop, and Xq22.3 Env (black lines) or Xq22.3 Env rev (dotted line) as control. Monoclonal antibody 13H5A5 was used as primary antibody.