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Fig. 1. Human ERK1 activated in vitro dimerizes in the presence of cellular cofactors. The highest activity is associated with bisphosphodimers. (A) Western blotting of recombinant hERK1 with anti-dualphosphorylated ERK antibody. (Lane 1) Unactivated protein. (Lane 2) Unactivated protein incubated in cell extract for 15 minutes. (Lane 3) hERK1 after a 1 hour activation by recombinant MEK, not incubated in cell extract. (Lane 4) hERK1 treated as for lane 3, but with an additional incubation in cell extract for 15 minutes. (Lane 5) hERK1 treated as for lane 4, followed by dephosphorylation using hCL100. (Lane 6) hERK1 treated as for lane 4, followed by additional activation by MEK overnight at 4°C. (Lane 7) hERK1 sample treated identically to the sample in lane 6, but dissolved in sample buffer containing 5% ß-mercaptoethanol. (Lane 8) Control: low ERK1 activity extract (30 minutes post-fertilization, 15 µg total cellular protein loaded); active ERK1 is undetectable. (-AB control) hERK1 sample treated as for lane 4, but primary antibody was omitted. The positions of molecular mass markers (kDa) are shown. (B) MAP kinase assays with myelin basic protein (MBP) using samples identical to those in panel A. Relative protein kinase activity is also shown from densitometry. (C) Western blotting using anti-ERK antibody of samples identical to those of lanes 2, 4, 5, 6 and -AB in panel A. The positions of molecular mass markers are shown. (D) Western blotting using anti-GST antibody of samples identical to lanes 4-6 and -AB in panel A. The positions of molecular mass markers are shown. (E) GST-{Delta}PEHD-hERK1, a dimerization-deficient mutant, does not show enhanced activity when incubated in whole-cell extract. 6 pM of each ERK1 recombinant protein was used per sample. Western blotting with anti-ERK antibody. (Lane 1) Unactivated {Delta}PEHD-hERK1. (Lane 2) {Delta}PEHD-hERK1 after a 1 hour activation by active MEK not incubated in cell extract. (Lane 3) {Delta}PEHD-hERK1 treated as for lane 2, but with an additional incubation in cell extract for 15 minutes. (Lane 4) {Delta}PEHD-hERK1 treated as for Lane 3, followed by additional activation by MEK overnight at 4°C. (Lane 5) An equal amount of recombinant control hERK1 treated as for lane 3. (F) MAP kinase assays with MBP using samples identical to those in E. Relative protein kinase activity is also shown from densitometry. Note that the monomeric mutant protein is effectively activated by MEK (eightfold over baseline); however, incubation in cell extract does not result in additional increase in its activity. The same amount of activated control wild-type protein forms a complex in cell extract with much higher kinase activity.





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