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Figure 2. Maspin and ß1 integrin coimmunoprecipitate and colocalize in MCF-10A cells grown in monolayers and in 3D culture. A) MCF-10A cells were chemically cross-linked, lysed, and 500 µg of protein extracts were immunoprecipitated with the indicated antibodies. An irrelevant rabbit antiserum was used as a negative control (lanes 3 and 6). Immunoprecipitates were separated in SDS-PAGE gels as detailed in Material and Methods and analyzed by Western blot as indicated. *Pre-ß1 integrin. B) MCF-10A cells were plated on coverslips, fixed, permeabilized, and stained with a mouse monoclonal antibody anti-human maspin (a) and a rabbit polyclonal anti-ß1 integrin (b). Nucleus is shown in blue (c) and merged image is shown in (d). Arrows indicate that maspin and ß1 integrin are located in the periphery of the cell (a and b, respectively) and are colocalized (d, Robs=0.704/Rrand=0.060±0.012); Bar 10 µm. C) MCF-10A cells were embedded in the Matrigel and allowed to form acini for 15 d. Cryosections (8 µm) were prepared and stained for maspin (a) and ß1 integrin (b), using the same antibodies mentioned above. Nuclei are shown in blue (c) and merged image is shown in (d). Arrow and arrowhead in (a) and (b) indicate localization on the basal membrane and in sites of cell–cell contact, respectively. Arrows in (d) indicate sites of maspin and ß1 integrin colocalization (Robs=0.580/Rrand=0.115±0.019); Bar 20 µm.
