Skip to main content
. 2005 Nov;25(22):9820–9828. doi: 10.1128/MCB.25.22.9820-9828.2005

FIG. 3.

FIG. 3.

Interaction of ERK5 with MEK5 isoforms. (A) Lysates (500 μg) of COS-7 cells expressing Flag-ERK5 were passed through a GST-MEK5α or a GST-MEK5β affinity chromatography column. Columns were washed twice prior to being eluted with increasing concentrations of KCl. The presence of ERK5 in the fractions was detected by immunoblot analysis using an anti-Flag antibody. (B) MEF extracts (500 μg) were incubated with bacterially expressed His-tagged ERK5 (1 μg). ERK5 was isolated by incubation with Ni-NTA agarose beads. The binding of MEK5 was examined by immunoblot analysis with an antibody to MEK5. (C) Lysate (250 μg) of COS-7 cells expressing HA-tagged ERK5 was incubated with 5 μg of GST, GST-tagged MEK5α, MEK5β, or α-Nter. MEK5 was isolated by incubation with GSH-agarose beads. The binding of ERK5 was examined by immunoblot analysis with an antibody to the HA epitope tag. (D) COS-7 cells were cotransfected with the expression vectors encoding Flag-ERK5 without (Cont) or with GST, MEK5α, or fragments of the N-terminal domain of MEK5α (amino acids 1 to 89, 1 to 69, and 1 to 29) fused to GST. Immunoblot analysis using an anti-Flag and an anti-GST antibody confirmed similar expression of ERK5 and MEK5 in cell lysates, respectively. ERK5 was isolated by incubation with anti-ERK5 antibody. The binding of MEK5 was examined by immunoblot analysis with an antibody to GST.