Comparison of membrane-associated- α_s1-casein solubilities in various detergents.

<p>A purified rough microsome fraction (A) or membrane-bound organelles from a PNS (B) were incubated under non-conservative conditions in the presence of saponin and centrifuged. The resulting membrane pellets were resuspended in HNE buffer in the absence (Control) or in the presence of the indicated detergents, and incubated for 30 minutes at 4°C. After centrifugation, supernatant (S) and pellet (P) were analysed via SDS-PAGE followed by either Coomassie blue staining (Protein staining) or immunoblotting (Immunoblot) with antibodies against either mouse milk proteins, Cnx or ERLIN2. Immature and mature α_s1-caseins were quantified by densitometry. For each condition, the amount of α_s1-casein recovered in the supernatant under the control condition was subtracted from that measured under other conditions, and the proportion of the immature or mature form in the pellet was expressed as percent of the total (sum of pellet and supernatant). The mean ± s.d. from four independent experiments is shown. Detergent-treated samples were compared to control two-by-two for either immature or mature α_s1-caseins using the Friedman’s test and statistical significance is indicated (*p<0.05). For Cnx and ERLIN2 representative immunoblots from two independent experiments are shown. Relative molecular masses (kDa) are indicated. im. α_s1-cas: immature α_s1-casein; m. α_s1-cas: mature α_s1-casein; TX-100: Triton X-100.</p

The DRMs containing α_s1-casein are sensitive to cholesterol depletion.

<p>Membrane-bound organelles in PNS or purified rough microsomes fractions were incubated in non-conservative buffer without Tween 20 and saponin, in the absence or the presence of the indicated concentration of mßCD for 30 minutes at 37°C. After centrifugation, supernatant (S) and pellet (P) were analysed via SDS-PAGE followed by immunoblotting with antibodies against mouse milk proteins or ERLIN2. For each type of membranes, three independent experiments are shown. The protein concentration in the analysis of the PNS 1 was twice lower than for all other samples and most of the scans showing α_s1-casein signal were taken from overexposed films for a better display of the large reduction of α_s1-casein present in the membrane pellet after cholesterol extraction by mßCD. Relative molecular masses (kDa) are indicated. im. α_s1-cas: immature α_s1-casein; m. α_s1-cas: mature α_s1-casein.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115903#pone-0115903-g005" target="_blank">Fig. 5 panel B</a>). 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.</p

A membrane-associated form of α_s1-casein is also present in the Golgi apparatus of rat MECs.

<p>(A) Time course for the arrival of newly synthesised caseins in the Golgi apparatus. Rat mammary gland fragments were pulse-labelled for 3 minutes with [³H]leucine and chased for the indicated times. At the end of the various chase periods, a PNS was prepared from the cells and analysed via SDS-PAGE and fluorography, followed by quantification of the immature (im.) and mature (m.) forms of both α_s1- and ß-casein (cas). The amount of the mature form of the caseins was expressed as percent of total (sum of immature and mature forms). The mean ± s.d. from three independent experiments is shown. (B) Relative proportions of membrane-associated forms of the caseins in the ER and the Golgi apparatus. Rat mammary gland fragments were either pulse-labelled for 3 minutes with [³H]leucine or pulse-labelled and chased for 5 minutes. Aliquots of the PNS prepared from the cells were subjected to centrifugation and the resulting membrane pellet was resuspended and incubated for 30 minutes in non-conservative buffer in the presence of saponin. After centrifugation, supernatants (S) and pellets (P) were analysed via SDS-PAGE and fluorography, followed by quantification of the immature (im.) and mature (m.) forms of both α_s1- and ß-casein. The amount of the mature form of the caseins (Table in panel B) was expressed as percent of total (sum of immature and mature forms). The amount of the various forms of the caseins in pellet (bar graph) is expressed as percent of total (sum of pellet and supernatant). The mean ± s.d. from three independent experiments is shown. Representative fluorograms are shown. Relative molecular masses (kDa) are indicated.</p

rVHSV expressing WNV E antigens induce IgG and neutralizing antibody production.

<p>(a) 5-week-old BALB/c mice in groups of 12 animals were immunized three times by subcutaneous (SC) injection.</p><p>(b) The titers of total IgG, IgG1 or IgG2a produced in immunized mice were determined by ELISA. The sera of 12 mice per group were harvested the day of the challenge (day 56) and pooled. The titer is the reciprocal of the last dilution at which the OD value measured is two times higher than the negative control (serum from day 0 for each group).</p><p>(c) The production of neutralizing antibodies was demonstrated by WNV neutralization assay. The titer of neutralizing antibodies is the reciprocal of the last dilution for which no CPE could be observed. Individuals with serum neutralization titers ≥ 30 are considered as positive.</p

rVHSV expressing WNV E antigens induce IgG and neutralizing antibody production.

<p>(a) 5-week-old BALB/c mice in groups of 12 animals were immunized three times by subcutaneous (SC) injection.</p><p>(b) The titers of total IgG, IgG1 or IgG2a produced in immunized mice were determined by ELISA. The sera of 12 mice per group were harvested the day of the challenge (day 56) and pooled. The titer is the reciprocal of the last dilution at which the OD value measured is two times higher than the negative control (serum from day 0 for each group).</p><p>(c) The production of neutralizing antibodies was demonstrated by WNV neutralization assay. The titer of neutralizing antibodies is the reciprocal of the last dilution for which no CPE could be observed. Individuals with serum neutralization titers ≥ 30 are considered as positive.</p

Appearance of the caseins in the Golgi region of lactating rat MECs.

<p>Mammary gland fragments from rat at mid-lactation were fixed and processed for electron microscopy. Golgi stacks, immature secretory vesicles (iSV) and other various distended elements of the Golgi region contain electron-dense particles loosely aggregated into interlaced structures or irregular linear clusters (arrows). These particles are also observed in distended rough ER components (see panel B, white arrowheads). Black arrowheads point to examples of close contact between electron-dense material and membranes of the compartments of the secretory pathway. Spherical compact casein micelles (CM) are found in mature secretory vesicles and in the lumen (Lu) of the acini (see panel B). N: nucleus; m: mitochondrion. Size of the bars is indicated.</p

Appearance of the caseins in immature and mature secretory vesicles.

<p>Mammary gland fragments from rat at mid-lactation were fixed and processed for electron microscopy. Large aggregates of electron-dense particles are found in immature secretory vesicles (see A–C) together with interlaced structures and irregular linear clusters (arrows). Spherical compact aggregates presenting the typical honeycombed texture of casein micelles (CM) are observed in mature secretory vesicles (see D–G). Arrowheads point to examples of close contact between the electron-dense material of the interlaced structures or casein micelles and the membranes of the secretory vesicles. ER: endoplasmic reticulum; m: mitochondrion. Size of the bars is indicated.</p

Analysis of virion incorporation of E_WNV antigens.

<p>Sucrose gradient-purified viral proteins were separated on a SDS-12% polyacrylamide gel. (A) Ten μg of total viral proteins were visualized after Coomassie blue staining. (B) Four μg (lanes 1 to 6) and 2 μg (lane 7) of total viral proteins were loaded. The gel was electrotransferred onto a nitrocellulose membrane and incubated with a mixture of mAb8150 and E24 anti-E_WNV antibodies. (C) Thirty μg of total viral proteins were visualized after Coomassie blue staining. Lane 8 corresponds to rE_VWN (1 μg was loaded on each gel).</p

Expression of E_WNV antigens in rVHSV-infected cells.

<p>The expression of E_WNV antigens was assessed by indirect-immunofluorescence in BF-2 cells. The cells were infected or not infected (Mock) either with the empty vector (rVHSV) or the six recombinant viruses expressing E_WNV domains (as indicated). Cells were incubated for 48 h at 14°C. (A) At 48 h post-infection, cells were fixed and permeabilized with a mixture of alcohol/acetone, and protein expression was detected using a monoclonal antibody against E_WNV DIII (E24). (B) Detection of membrane expression of E_WNV antigens was performed on live cells using the E24 antibody, except for rVHSV-SP_GDIIDIII_WNVTM_G infected cells where E_WNV expression was achieved with mAb8150 (magnification X63).</p