Colocalization of TGFβ
integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ1 (from R&D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction , slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope . Magnification 1000 ×.