Co-immunoprecipitation analyses to detect MAL2/MUC1 interactions in Triton X-100-soluble versus-insoluble fractions. MCF-10A/Myc-MAL2 cells were extracted with 1% Triton X-100 at 4°C, and subjected to sucrose gradient centrifugation. Antisera employed in Western blot analyses are shown at the left, and sizes of detected proteins are shown at the right. (a) Fractions (Fr, as shown above the top panel) of 1 ml were collected, and aliquots from each were subjected to SDS-PAGE and Western blot analysis with MUC1 and MAL2 antisera. Vertical lines between fractions 8 and 9 distinguish samples loaded on different gels. (b) Pooled fractions (fractions 1–4, which include lipid rafts, fractions 5–8 and fractions 9–12) were immunoprecipitated with MUC1 or MAL2 antisera either alone or with MAL2 peptides (+Pep), as shown at the top of the panel. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with MUC1 monoclonal antibody. Results shown represent those obtained from 3 independent experiments.
Fanayan et al. BMC Cell Biology 2009 10:7 doi:10.1186/1471-2121-10-7