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. 2005 Oct;25(20):8960–8970. doi: 10.1128/MCB.25.20.8960-8970.2005

FIG. 5.

FIG. 5.

Hey proteins downregulate the ANF promoter in vitro and they interact with GATA proteins. (A) GATA4/6-driven ANF expression is repressed by Hey1, Hey2, and HeyL. HEK293 cells were transfected with the ANF-luciferase reporter together with GATA4 and GATA6 and the different Hey1, Hey2, and HeyL expression plasmids. Basal activity with empty pCS2 vector was set to 100%. GATA4 and -6 activate the ANF promoter, which is completely blocked by Hey proteins (B) In HEK293 cells, basal ANF promoter activity is repressed by Hey1, Hey2, and to a lesser extent by HeyL. (C) Hey proteins interact with GATA4 in mammalian cells. HEK293T cells were cotransfected with HA-GATA4 and either FLAG-Hey1, -Hey2, -HeyL, or empty pCS2-FLAG expression vector. After 40 h cells were lysed and protein expression was verified by Western blotting (left). Lysates were incubated with an anti-HA antibody linked to CNBr-Sepharose to pull down HA-GATA4 and interacting proteins. After washing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoprecipitates were probed with anti-FLAG antibody to identify interacting FLAG-tagged Hey proteins. This interaction is specific, as no FLAG-Hey1 could be detected in the absence of HA-GATA4. (D) Hey proteins interact with GATA6. FLAG-GATA6 was pulled down from lysates of cotransfected cells with an anti-FLAG antibody bound to protein A-Sepharose. Protein interaction with HA-tagged Hey proteins is detected with an anti-HA antibody.