ORMDL fused to EGFP at the amino or carboxyl terminus localizes in the perinuclear ER and throughout the ER network. (a-c) Confocal scanning microscopy of COS-7 cells transfected with ORMDL1-EGFP (a) co-localize with protein-disulfide isomerase (PDI) (b), an ER-marker protein, showing an overlapping signal in the endoplasmic reticulum (c). (d-f) Double-label fluorescence with biotin-labeled concanavalin A showed no overlapping signal with ORMDL1-EGFP at the plasma membrane. (g-l) COS-7 cells transfected with EGFP-ORMDL1, EGFP-ORMDL2 and EGFP-ORMDL3 fusion proteins were similar either in fixed (g, h, and i, respectively) and in vivo (j) cells, co-localizing with an in vivo ER marker (k). EGFP alone was uniformly distributed throughout the cell, including the nucleus (l).