Our previous study on milk caseins. Second, non covalent interactions have also been observed in this compartment for the immature ER form of as1-casein which is not phosphorylated. In contrast, immature -casein is poorly aggregated in the ER. Finally, our experiments reveal the existence of a membrane-associated form of as1-casein within the secretory pathway of 
MEC. We 
have found that protein dimerization via the disulphide bond greatly potentialize this interaction of as1-casein with the membranes. Of note, bovine as1-casein, which does not contain cysteine, is also known to dimerize at physiological ionic strength and pH. Since as1-casein is required for the efficient export of the other caseins from the ER, we believe that its membranous form may play a key role in the early steps of casein micelle biogenesis and/or casein transport in the secretory pathway. This conclusion is also supported by the observation that modification of the relative proportion of as1-casein due to low or lack of HJC0350 site expression invokes the accumulation of the other caseins in the ER coupled to an ER stress response, notably signalled by an enhanced expression of ER-resident proteins. In the present study, we further investigate the molecular basis of as1-casein association with MLi-2 web membranes of the secretory pathway, with emphasis on its potential incorporation into detergent-resistant membranes microdomains, an interaction which may be a prerequisite for the formation, transport and sorting of casein micelle to the apical plasma membrane domain of the MEC. 3 / 25 Membrane-Associated as1-Casein Binds to Cholesterol-Rich Microdomains Materials and Methods Animals and antibodies Wistar rats raised in our institute were euthanatized by decapitation at mid-lactation. Animal welfare and conditions for animal handling were in accordance with the guideline of the European Community for experimental animal use and experiments were approved by the French Ministry of Agriculture and Forestry and the Ministry of Research and Technology. E. Chanat owns an accreditation for animal experimentation, level 1. Antibodies against mouse whole milk proteins were obtained from Nordic Immunological laboratories and used at a dilution of 1:2500 for immunoblotting. Rabbit polyclonal antibodies against calnexin were purchased from StressGen Biotechnologies Corp. and used at a 1:1000 dilution. Rabbit polyclonal antibodies against ERLIN2 were from Sigma and used at 1:2500 dilution. Mouse monoclonal antibody to protein disulfide isomerase was from Enzo Life Sciences and used at 1:5000 dilution. HRP-conjugated secondary antibodies were obtained from goat or from sheep and used at a dilution of 1:5000 or 1:2000, respectively. Unless otherwise indicated, chemicals were obtained from SigmaAldrich or Research Organics. Metabolic labelling Metabolic labelling of mammary fragments was performed essentially as previously described, except that leucine was used. Briefly, dams were euthanatized and mammary glands were excised bilaterally and transferred to icecold 0.25 M sucrose. Samples were dissected from the connective tissue and muscles, finely chopped into <12 mm3 pieces using scissors on an ice-cold plastic pad, and further minced using a homemade multi-mounted razor blade device. Fragments were preincubated for 30 minutes in PubMed ID:http://jpet.aspetjournals.org/content/120/3/269 leucine-free DMEM under an atmosphere of 95 O2/5 CO2, and then pulse-labelled for 3 minutes in a small volume of leucine-free DMEM containing 1.85 MBq/ ml L-leucine, al.Our previous study on milk caseins. Second, non covalent interactions have also been observed in this compartment for the immature ER form of as1-casein which is not phosphorylated. In contrast, immature -casein is poorly aggregated in the ER. Finally, our experiments reveal the existence of a membrane-associated form of as1-casein within the secretory pathway of MEC. We have found that protein dimerization via the disulphide bond greatly potentialize this interaction of as1-casein with the membranes. Of note, bovine as1-casein, which does not contain cysteine, is also known to dimerize at physiological ionic strength and pH. Since as1-casein is required for the efficient export of the other caseins from the ER, we believe that its membranous form may play a key role in the early steps of casein micelle biogenesis and/or casein transport in the secretory pathway. This conclusion is also supported by the observation that modification of the relative proportion of as1-casein due to low or lack of expression invokes the accumulation of the other caseins in the ER coupled to an ER stress response, notably signalled by an enhanced expression of ER-resident proteins. In the present study, we further investigate the molecular basis of as1-casein association with membranes of the secretory pathway, with emphasis on its potential incorporation into detergent-resistant membranes microdomains, an interaction which may be a prerequisite for the formation, transport and sorting of casein micelle to the apical plasma membrane domain of the MEC. 3 / 25 Membrane-Associated as1-Casein Binds to Cholesterol-Rich Microdomains Materials and Methods Animals and antibodies Wistar rats raised in our institute were euthanatized by decapitation at mid-lactation. Animal welfare and conditions for animal handling were in accordance with the guideline of the European Community for experimental animal use and experiments were approved by the French Ministry of Agriculture and Forestry and the Ministry of Research and Technology. E. Chanat owns an accreditation for animal experimentation, level 1. Antibodies against mouse whole milk proteins were obtained from Nordic Immunological laboratories and used at a dilution of 1:2500 for immunoblotting. Rabbit polyclonal antibodies against calnexin were purchased from StressGen Biotechnologies Corp. and used at a 1:1000 dilution. Rabbit polyclonal antibodies against ERLIN2 were from Sigma and used at 1:2500 dilution. Mouse monoclonal antibody to protein disulfide isomerase was from Enzo Life Sciences and used at 1:5000 dilution. HRP-conjugated secondary antibodies were obtained from goat or from sheep and used at a dilution of 1:5000 or 1:2000, respectively. Unless otherwise indicated, chemicals were obtained from SigmaAldrich or Research Organics. Metabolic labelling Metabolic labelling of mammary fragments was performed essentially as previously described, except that leucine was used. Briefly, dams were euthanatized and mammary glands were excised bilaterally and transferred to icecold 0.25 M sucrose. Samples were dissected from the connective tissue and muscles, finely chopped into <12 mm3 pieces using scissors on an ice-cold plastic pad, and further minced using a homemade multi-mounted razor blade device. Fragments were preincubated for 30 minutes in PubMed ID:http://jpet.aspetjournals.org/content/120/3/269 leucine-free DMEM under an atmosphere of 95 O2/5 CO2, and then pulse-labelled for 3 minutes in a small volume of leucine-free DMEM containing 1.85 MBq/ ml L-leucine, al.
