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. 2006 Mar;26(6):2065–2079. doi: 10.1128/MCB.26.6.2065-2079.2006

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Copyright © 2006, American Society for Microbiology

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FIG. 1. — PB1 domain-dependent interaction of MEK5 and ERK5. (A) The indicated MEK5 GST fusion proteins were tested for their ability to bind ERK5. Xpress-ERK5 from HEK293 cell lysates was used in pull-down assays using GST fusions of the MEK5 PB1 domain (residues 18 to 97), MEK5 residues 98 to 165, MEK5 residues 18 to 165 lacking the kinase domain (Δkinase), the MEK5 kinase domain (residues 166 to 448), or a control using the MEKK2 PB1 domain. Adsorbents were resolved by SDS-PAGE and immunoblotted with anti-Xpress antibody. The amount of GST fusion protein used in each assay is shown by Coomassie blue staining (bottom panel). (B) In vivo binding of MEK5 protein sequences and ERK5. Full-length Xpress-ERK5 was expressed with FLAG-MEK5 deletion constructs. OSM (for osomsensing scaffold for MEKK3), which does not bind MEK5, was used as a control. FLAG-proteins were immunoprecipitated with anti-FLAG antibody. The adsorbents were analyzed by immunoblotting with anti-Xpress or anti-FLAG antibody (left panel). The expression of each protein was confirmed by immunoblotting of whole-cell lysates (right panel). (C) Demonstration of a MEKK2-MEK5-ERK5 ternary complex in cells. FLAG-tagged WT, Δkinase, and ³¹¹S³¹⁵T-AV MEK5 (^311.5ST-AV in the figure) proteins were expressed with HA-tagged MEKK2 and Xpress-tagged ERK5. HA-tagged MEKK2 was immunoprecipitated with anti-MEKK2 antibody, and the immunocomplexes were analyzed by using anti-Xpress (for ERK5) and anti-FLAG (for MEK5) antibody immunoblots (left panel). The expression of each protein was confirmed by anti-MEKK2, anti-FLAG, and anti-Xpress antibody immunoblotting of whole-cell lysates (right panel). (D) Specificity of the MEK5 Δkinase(18-165) protein interaction with MAPKs. Xpress-ERK5, FLAG-p38, HA-JNK, HA-ERK1, or HA-MEKK2 were tested for binding of the GST-MEK5 Δkinase(18-165) fusion protein. The adsorbents were resolved and sequentially immunoblotted with anti-Xpress (for ERK5), anti-FLAG (for p38), and anti-HA (MEKK2, JNK, and ERK1) antibodies (left panel). Protein expression for each construct is shown by blotting of whole-cell lysates (right panel). Nonspecific bands in the anti-HA blots are marked with an asterisk.