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Fig. 8. G3EGF expression promoted EGFR/integrin association. (A) Maintained in different conditions as indicated, U87 cells were lysed and subjected to immunoprecipitation with anti-EGFR antibody, followed by western blot analysis using an anti-ß₁-integrin antibody as probe. In the presence of serum, ß₁ integrin was precipitated with EGFR. (B) Jurkat cells were cultured in DMEM containing 10% FBS for 4 days. Approximately 20% of the cells attached to the tissue culture plates (adh) and 80% remained in suspension (susp). Both populations of cells were lysed separately with equal amounts of lysis buffer and subjected to immunoprecipitation with anti-EGFR antibody followed by western blot analysis, probed with anti-ß₁-integrin antibody. Cell lysate was also analysed by western blotting probed with antibodies against ß₁-integrin or EGFR to assess their individual protein levels. Even with lower integrin protein levels (middle), the adherent cultures had higher levels of integrin-EGFR interaction (left). (C) Cell cycles were analysed on the two populations of Jurkat cells. The adherent cells had a larger proportion of cells entering into the S phase (45%) than those in suspension, implying a greater rate of proliferation. (D) G3EGF- and vector-transfected U87 cells were maintained in DMEM containing 10% FBS for 24 hours, followed by serum withdrawal and incubation in serum-free DMEM for different times. Cell lysate was prepared and subjected to immunoprecipitation with anti-EGFR antibody, followed by western blotting probed with anti-ß₁-integrin antibody. Expression of G3EGF enhanced the association of EGFR and ß₁-integrin before serum withdrawal and delayed the disassociation of EGFR and ß₁-integrin after serum withdrawal. Cell lysate was also analysed by western blotting probed with anti-ß₁-integrin antibody or anti-EGFR antibody to assess the expression of these two molecules. All experiments were repeated at least three times.

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