Fig. 8. ERGIC-53 is present in a complex of high density, irrespective of the presence or absence of intermolecular disulfide bonds. (A) Separation of soluble and membrane-bound marker proteins by rate-zonal centrifugation. Shown is a representative sucrose-density gradient with the position of marker proteins in kDa. The molecular sizes of the membrane proteins CD4 (48 kDa) and sucrase-isomaltase (209 kDa) are in italics. (B) Analysis of sucrose density gradient fractions by SDS-PAGE/fluorography. Lec-1 cells stably expressing GM were pulse-labeled with [³⁵S]-methionine for 5 minutes. After a 60 minute chase, the cells were lysed and cleared lysates were fractionated by sucrose gradient centrifugation. Fractions were immunoprecipitated with anti-myc and immunoprecipitates were separated by nonreducing SDS 4-10% PAGE followed by fluorography. Arrows indicate dimeric (2x) and hexameric (6x) forms. (C) Fractionation of GM with C₄₆₆A/C₄₇₅A substitution (fluorogram of a 7% SDS gel). The experiment was performed as in panel B but with Lec-1 cells stably expressing a GM variant, with C₄₆₆ and C₄₇₅ mutated to alanines. 1x, monomeric form.