Intracellular retention of AICL is an inherent property of the lectin-like domain. (A) Schemes of the AICL/KACL hybrids with calculated molecular masses (deglycosylated proteins). AICL domains are shown in gray, KACL domains are open. Expression of hybrid proteins was analyzed 3 days after transfection of 293 cells. (B) Lysates of transfected 293 cells were left untreated (ø), or treated with endoglycosidase H or PNGase F, separated by SDS-PAGE, and KACL, AICL, and hybrid proteins detected using anti–FLAG-tag mAb M2 by immunoblotting. (C) Relative surface expression of AICL, KACL, and AICL/KACL hybrids by transfected 293 cells. Transfectants were stained with mAb M2 and mean fluorescence intensities (MFI) determined by flow cytometry with values set in relation to the MFI of AICL transfectants (arbitrarily set as 1) (left). Representative histograms of hybrids 2 and 4 (solid, dark gray) overlayed with those of AICL (thin line) and KACL (thick line) transfectants, and untransfected 293 cells (solid, light gray) (right). One representative experiment out of 3 is shown. (D) Confocal microscopy analysis of 293 cells ectopically expressing AICL, KACL, or hybrid 2, respectively. Transfectants were costained with mAb M2 (green) and anti-Giantin (red). Scale bars represent 10 µm.