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Fig. 1. {alpha}4ß1- and {alpha}6ß1-integrins bind to MK. (A) The binding of ß1-integrin to MK. The cell lysate of the COS-7 cells transfected with ß1-HA-encoding cDNA was applied to an MK-agarose column (0.2 ml) and bound proteins were eluted stepwise with 1 ml of buffer containing 0.15 M, 0.2 M, 0.3 M, 0.4 M and 0.5 M NaCl with or without 20 mM EDTA. A portion of the eluate was subjected to SDS-PAGE and analysed by western blotting using anti-HA antibody to detect ß1-integrin. (B) Identification of {alpha}-subunit of ß1-integrin capable of binding MK. The lysate of COS-7 cells transfected with ß1-HA was applied to the MK column and eluted with 0.5 M NaCl containing EDTA. The eluate was analysed by immunoblotting using anti-{alpha}4-integrin or anti-{alpha}6integrin antibodies. (C) Binding of {alpha}4-integrin to MK. The cell lysate of COS-7 cells transfected with FLAG-tagged {alpha}4-integrin-encoding cDNA was applied to the MK column before or after affinity purification using anti-FLAG-antibody/agarose, and bound proteins were eluted and analysed as in A, except that anti-FLAG antibody was used. (Before) MK column only. (After) MK column and FLAG column. (D) Binding of {alpha}6-integrin to MK. The cell lysate of COS-7 cells transfected with HA-tagged {alpha}6-integrin-encoding cDNA was applied to the MK column before or after affinity purification using laminin-agarose, and bound proteins were eluted and analysed as in A. (Before) MK column only. (After) MK column and FLAG column. (E) Binding of metabolically labeled {alpha}4ß1-integrin to MK. (Flag) COS-7 cells transfected with FLAG-tagged {alpha}4-integrin cDNA were labeled with [35S]-methionine. After purification using anti-FLAG-antibody/agarose, the radioactively labeled {alpha}4ß1-integrin fraction, which was bound to the MK-agarose column and was eluted by 0.5 M NaCl with 20 mM EDTA, and analysed by SDS-PAGE. (HA) COS-7 cells were transfected with FLAG-tagged {alpha}4-integrin and HA-tagged ß1-integrin-encoding cDNAs. After purification using anti-HA-antibody/agarose, the radioactively labeled {alpha}4ß1-integrin fraction, which was bound to the MK-agarose column, was analysed as in the case of `FLAG'.