Fig. 1. GFP-ERGIC-53 localizes and oligomerizes like endogenous ERGIC-53. (A) HeLa cells stably expressing GFP-ERGIC-53 were treated with sodium butyrate overnight, fixed with paraformaldehyde, and stained for ß-COP, Sec31 or transferrin receptor. Cells were observed with a confocal fluorescence microscope. Insets show higher magnifications. (B) GFP-ERGIC-53 cells treated with sodium butyrate or non-transfected cells were pulsed with [³⁵S]methionine and chased for the indicated times. Proteins were immunoprecipitated either with anti-ERGIC-53 for the control cells or with anti-GFP for the stable clone. Immunoprecipitates were analyzed by non-reducing SDS-PAGE and visualized by fluorography. During the time course, dimers (lower bands) disappear as hexamers (higher bands) form until a steady state is reached when each species is represented as 50%. GFP-ERGIC-53 dimers appear as two bands corresponding to homodimers and heterodimers (lower arrowhead) with endogenous ERGIC-53. These dimers are converted to hetero-hexamers (upper arrowheads) and homo-hexamers. (C) Quantification of dimer and hexamer formation during the chase period. 100% is the sum of homo-dimers and homo-hexamers at a given chase time. A representative experiment is shown. Bar, 5 µm.