sERGIC-53 binds to HeLaS3 cells in a MCFD2 and CaCl₂-dependent manner. (A) The domain structures of ERGIC-53 (top) and MCFD2 (bottom). SS indicates signal sequence; Lectin, lectin domain; stalk, stalk domain; TM, transmembrane domain; C, cytoplasmic domain; EF, EF-hand domain. (B) Purified recombinant sERGIC-53 and MCFD2 (shown in gray in panel A) were electrophoresed in a 12.5% polyacrylamide gel under reducing conditions and stained with Coomassie brilliant blue. Molecular weight markers are indicated on the left. (C) HeLaS3 cells were incubated with 20 μg/mL sERGIC-53-SA (left column) or sERGIC-53-SA and MCFD2 complexes at a molar ratio of 1:4 (sERGIC-53-SA-MCFD2, right column), in the presence of 1 mM CaCl₂ or 1 mM EDTA, and then analyzed by flow cytometry. MFI, mean fluorescence intensity. (D) 20 μg/mL sERGIC-53-SA was mixed with MCFD2 at ratios of 1:0.25, 0.5, 0.75,1, 2, 3, 4, or 5, and then added to the cells. The binding was monitored by flow cytometry. (E) 10 μg/mL sERGIC-53-SA was preincubated with wild-type (wt) MCFD2 or the D129E and I136T mutants at a ratio of 1:4 before being added to HeLaS3 cells and subjected to flow cytometric analysis. Filled histograms indicate the cells bound to sERGIC-53-SA or sERGIC-53-SA-MCFD2. Thin lines show the background staining of PE-SA. The numbers in each panel indicate the MFI. Data are representative of 3 independent experiments with similar results.