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. 2004 Jun;15(6):2794–2803. doi: 10.1091/mbc.E03-12-0879

Figure 1.

Figure 1.

ERK/MLCK activities are required for Src-induced integrin adhesion assembly in KM12C colon cancer cells. (A) KM12C cells transfected with Src527F (KM12C/Src527F) were cultured in uncoated plastic dishes (adherent) or in poly-HEMA–coated dishes (suspension). Phospho-ERK or phospho-MLC levels were detected by probing total lysates with the anti-phospho-ERK (Thr202/Tyr204) or anti-phospho-MLC (Ser19) antibodies (top panels). The filters were reprobed with anti-ERK or anti-MLC antibodies (lower panels). (B) Increased level of total cellular phospho-MLC in KM12C/Src527F cells was blocked by MLCK inhibitor ML9 (7.6 μM). (C) (a–f) KM12C/Src527F (a–c) or KM12C/vector (d–f) cells were plated on fibronectin-coated substratum for 6 h, fixed, and stained with anti-paxillin (a and d), anti-phospho-ERK (Thr202/Tyr204; b and e) or anti-phospho-MLC (Ser19; c and f) antibodies. Arrows show paxillin, phospho-ERK and phospho-MLC localized at cell-matrix adhesion complexes at the ends of protrusive structures in KM12C/Src527F cells. (g–m) MEK inhibitor UO126 (25 μM; g–i) or MLCK inhibitor ML7 (5 μM; j–m) blocked the formation of protrusive integrin-mediated adhesions in KM12C/Src527F cells. Arrows show localization of paxillin or phospho-ERK at nonprotrusive cell-matrix adhesion structures at the cell periphery. Scale bars, 10 μm. (D) KM12C/Src527F cells were plated on poly-l-lysine–coated substratum, fixed, and stained with anti-paxillin (a), anti-phospho-ERK (Thr202/Tyr204; b) or anti-phospho-MLC (Ser19; c) antibodies. Scale bars, 10 μm.