Figure 6. Membrane-associated-α_s1-casein is associated with DRMs.

A purified rough microsome fraction (A) or membrane-bound organelles from a PNS (B) were incubated in the absence of saponin (−Saponin) or under non-conservative conditions in the presence of saponin (+Saponin) and centrifuged. Supernatant was removed and membrane pellets were resuspended in HNE buffer, in the absence (Control) or the presence of the indicated detergents, and incubated for 30 minutes at 4°C. Detergent-treated membranes were subjected to flotation on a sucrose step gradient (sucrose concentrations as indicated in Fig. 5 panel B). Half of the supernatant (Sup), fractions collected from top to bottom (1–5) and gradient pellet (P) were analysed via SDS-PAGE followed by immunoblotting with an antibody against mouse milk proteins. Representative ECL signals from four experiments with three independent organelles preparations are shown. The distribution of ERLIN2 was analysed within the immunoblots shown in panel A. C. Quantification of membrane-associated-α_s1-casein in DRMs. Immature (Microsomes), or immature and mature α_s1-caseins (PNS) were quantified via densitometry. For each condition, the amounts of the indicated forms of α_s1-casein recovered in the various fractions of the sucrose step gradient were measured and the proportion of the immature or mature forms of α_s1-casein for each fraction was expressed as percent of the total (sum of gradient fractions and pellet). The means ± s.d. from four experiments with three independent organelles preparations are shown. The proportion of either immature or mature α_s1-caseins in each fraction of the gradient from TX-100-treated samples was compared two-by-two to control data using the Friedman’s test and statistical significance is indicated (*p<0.05). Relative molecular masses (kDa) are indicated. im. α_s1-cas: immature α_s1-casein; m. α_s1-cas: mature α_s1-casein; im. ß-cas: immature ß-casein; m. ß-cas: mature ß-casein; TX-100: Triton X-100; *: protein band with electrophoretic mobility identical to PDI. F: fraction; TX-100: Triton X-100.