(A) HeLa cells were serum-starved for 6 h and stimulated with saline (a–c) or 10 ng/ml EGF (d–f) for 10 min. Cells were fixed with 4% formaldehyde and stained with an anti-pERK1/2 antibody (a and d) or an anti-GIT1 antibody (b and e). Panels c and fare the merged images. (B) HeLa cells were serum-starved for 6 h and stimulated with EGF (10 ng/ml) for the times indicated. Cytoskeleton fractions were immunoprecipitated with an anti-GIT1 antibody and probed with an anti-ERK1/2 antibody (top panel). To confirm equal protein loading, the blot was reprobed with the anti-GIT1 antibody (bottom panel). These results were reproducibly obtained in three independent experiments. IB, immunoblot; IP, immunoprecipitation.

(A) ERK2 sequences were analysed using the Lupas method and one major CC domain (amino acids 322–343) was found to exist in the C-terminus of ERK2. (B) HeLa cells were co-transfected with Xpress–GIT1 and FLAG–ERK2 or Xpress–GIT1 and FLAG–ERK2(del CC) for 24 h, serum-starved for 6 h, and then stimulated with EGF as indicated. The cell cytoskeletons were separated as described in the Materials and methods section. Cell cytoskeleton fractions were immunoprecipitated with an anti-Xpress antibody and probed with an anti-FLAG antibody (top panel). To confirm equal protein loading, the blot was re-probed with an anti-Xpress antibody (middle panel). Protein expression was detected by probing with an anti-FLAG antibody in cytoskeleton fractions (bottom panel). (C) GST–GIT1 was immobilized on glutathione-conjugated beads and incubated with cytoskeleton fractions from FLAG–ERK2- or FLAG–ERK2(del CC)-transfected HeLa cells. Beads were washed extensively and then immunoblotted for FLAG–ERK2 (top panel) and reprobed with an anti-GST antibody to confirm equal loading (middle panel). To confirm equal protein expression, cytoskeleton fractions were blotted with an anti-FLAG antibody (bottom panel). (D) HeLa cells were co-transfected with HA–MEK1 and FLAG–ERK2 or HA–MEK1 and FLAG–ERK2(del CC) for 24 h, serum-starved for 6 h, and then stimulated with EGF as indicated. The cell cytoskeletons were separated as described in the Materials and methods section. Cell cytoskeleton fractions were immunoprecipitated with an anti-HA antibody and probed with an anti-FLAG antibody (top panel). To confirm equal protein loading, the blot was re-probed with an anti-HA antibody (bottom panel). These results were reproducibly obtained in three independent experiments. IB, immunoblot; IP, immunoprecipitation.

(a) HeLa cells were transfected with pcDNA3 or FLAG–ERK2(del CC) for 24 h and resuspended in DMEM with 0.5 µg/ml trypsin inhibitor. Cells were washed three times and seeded on to fibronectin coated-dishes (10 µg/ml) and cell spreading was measured at 10 min. Cells were fixed with 4% formaldehyde. Then, 100 attached cells were randomly selected to measure the cell area using ImageJ software (*P<0.01; mean±S.E.M.; n=6). The cell area for pcDNA3 was arbitrarily set to 100. (b) HeLa cells transfected with pcDNA3 or FLAG–ERK2(del CC) for 24 h, were serum-starved for 6 h and then seeded in the upper portion of a Boyden chamber on collagen precoated PVP-free polycarbonate membranes. EGF was added to the lower chamber. Cells were incubated for 6 h at 37 °C in a 5% CO₂ humidified incubator. The membranes were removed and cells were stained. The relative increases in cell density were determined by quantitative densitometry (*P<0.01; mean±S.E.M.; n=6).

HeLa cells were co-transfected with Xpress–GIT1 and either FLAG–ERK2(del 10–25) or Xpress–GIT1 with FLAG–ERK2(1–343) for 24 h, serum-starved for 6 h, and then stimulated with EGF as indicated. The cell membrane, cytoplasm and cytoskeleton were separated as described in the Materials and methods section. Cytoskeletal fractions were immunoprecipitated with an anti-Xpress antibody and probed with an anti-FLAG antibody (top panel). To confirm equal protein immunoprecipitation, the blot was reprobed with an anti-Xpress antibody (bottom panel). These results were reproducibly obtained in three independent experiments. IB, immunoblot; IP, immunoprecipitation.

(A) HeLa cells were transfected with pcDNA3 (a and b) or FLAG–ERK2(del CC) (c and d) for 24 h, serum-starved for 6 h, and then stimulated with EGF as indicated. Cells were fixed with 4% formaldehyde and stained with an anti-pERK1/2 antibody. (B) HeLa cells were transfected with pcDNA3 (a, b and c) or FLAG–ERK2(del CC) (d, e and f) for 24 h, serum-starved for 6 h, and then stimulated with EGF for 10 min. Cells were fixed with 4% formaldehyde and stained with an anti-pERK1/2 antibody (a and d) or anti-GIT1 antibody (b and e). Panels c and f are the merged images of a and b, and d and e respectively. These results were reproducibly obtained in three independent experiments.