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. Author manuscript; available in PMC: 2010 May 12.
Published in final edited form as: Mol Cancer Res. 2009 May 12;7(5):634–644. doi: 10.1158/1541-7786.MCR-08-0371

Figure 6.

Figure 6

Syk is activated by integrin-crosslinking in MDA-MB-231 cells. A, after being transiently transfected to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD-Syk), MDA-MB-231 cells were removed and plated onto fibronectin-coated coverslips. After different time points (0, 10, 30, and 60 min), cell lysates were collected and analyzed by Western blotting with antiphosphotyrosine antibodies. The migration positions of Syk-EGFP or Syk-EGFP(K396R) are indicated by the arrowheads. B, integrins were crosslinked for the indicated times (in minutes) with anti-β1 integrin antibodies on MDA-MB-231 cells in suspension that were transiently expressing Syk-EGFP (Syk), Syk-EGFP(Y317F/Y342F/Y346F) (F3), EGFP (EGFP) or Syk-EGFP(K396R) (KD-Syk). Cell lysates were analyzed by Western blotting with antibodies against phosphotyrosine (pY), Syk or GAPDH. The migration positions of Syk-EGFP or Syk-EGFP mutants are indicated by the closed arrowheads and that of GAPDH by the open arrowheads. C, integrins were crosslinked for the indicated times (in minutes) with anti-β1 integrin antibodies on MDA-MB-231 cells in suspension that were transiently expressing Syk-EGFP(Y317F) (Y317F), Syk-EGFP(Y342F/Y346F) (Y342F/Y346F), Syk-EGFP (Syk), Syk-EGFP(Y342F) (Y342F) or Syk-EGFP(Y346F) (Y346F). Cell lysates were analyzed by Western blotting with antibodies against phosphotyrosine (pY), Syk or GAPDH. The migration positions of Syk-EGFP or Syk-EGFP mutants are indicated by the closed arrowheads and that of GAPDH by the open arrowheads. D, The ability of MDA-MB-231 cells transfected with expression plasmids for Syk-EGFP (Syk), Syk-EGFP(K396R) (KD-Syk), Syk-EGFP(Y317F/Y342F/Y346F) (F3), Syk-EGFP(Y317F) (Y317F), Syk-EGFP(Y342F/Y346F) (F2), Syk-EGFP(Y342F) (Y342F) or Syk-EGFP(Y346F) (Y346F) to migrate through the pores of a transwell filter was measured. Motility was normalized to that of cells expressing Syk-EGFP. The data shown represent the mean and standard deviations of three independent experiments. * P < 0.05.