Figure 5. CIP interacts with Isl1 and represses its transcription activity.
(A) Co-immunoprecipitation assays showing the interaction between Flag-tagged CIP and Myc-tagged Isl1. HEK-293T cells were co-transfected Flag-CIP, Myc-Isl1, or both. Anti-Flag antibody was used to immunoprecipitate (IP) cell extracts and anti-Myc antibody was used in Western blot (WB) to detect Myc-Isl1 protein in the complex. 10% cell lysate was used as input to demonstrate the expression of tagged proteins (button two panels).
(B) In vitro GST-fusion protein pull down assays showing the direct interaction between CIP and Isl1. Isl1 protein was synthesized in vitro and labeled with S-35. GST-CIP protein, but not GST protein, pulled down S-35 labeled Isl1. 20% of labeled Isl1 protein was load as control (Input).
(C) Map the interaction domains of the CIP protein that mediate the interaction between Isl1 and CIP using co-immunoprecipitation assays. HEK-293T cells were co-transfected Flag-Isl1, Myc-CIP full-length (1-309) or indicated truncated mutants, or both. Anti-Flag antibody was used to immunoprecipitate (IP) cell extracts and anti-Myc antibody was used in Western blot (WB) to detect Myc-CIP protein in the complex. 10% cell lysate was used as input to demonstrate the expression of tagged proteins (lower two panels). The interaction domains are summarized in the bottom diagram.
(D) Map the interaction domains of Isl1 proteins which mediate the interaction between Isl1 and CIP using co-immunoprecipitation assays. HEK-293T cells were co-transfected Myc-CIP, Flag-Isl1 full-length (1-349) or indicated truncated mutants, or both. Anti-Flag antibody was used to immunoprecipitate (IP) cell extracts and anti-Myc antibody was used in Western blot (WB) to detect Myc-CIP protein in the complex. 10% cell lysate was used as input to demonstrate the expression of tagged proteins (lower two panels). The interaction domains are summarized in the bottom diagram.
(E) Isl1 and CIP are co-expressed in cardiomyocytes of outflow tract of mouse embryos. Immunohistochemistry to demonstrate that Isl1 (green) and β-gal positive CIP (red) expressing cells are co-located in the cardiomyocytes of outflow tract (OFT) of E10.5 mouse embryos (arrowheads). DAPI staining marks the nucleus. Bar = 50 μm. A, atrium; LV, left ventricle; OFT, outflow tract.
(F) Enlargement of boxed area in (E) to show that Isl1 and CIP expression is overlapped in the OFT cardiomyocytes (white arrowheads). The expression of Isl1 (green), CIP (as marked by β-gal positive cells, red) DAPI (blue) and merged images were shown. Isl1 and CIP positive cells were indicated by white arrowheads. Note that Isl1 is not expressed in the cardiomyocytes of atrium where CIP is expressed (yellow arrows). (G) CIP is a transcriptional repressor. HEK293T cells were transiently transfected with expression vectors encoding the CIP fused to GAL4 (1–147) and the pL8G5-luciferase reporter, which contains binding sites for the GAL4 DNA binding domain. Luciferase activity is expressed as relative activity in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in triplicate. *P<0.05.
(H) CIP represses the transcription activity of Isl1. MEF2C enhancer-luciferase reporter was co-transfected with indicated expression plasmids in HEK293T cells and luciferase activity was measured. Results were presented as relative luciferase activity in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in triplicate. *P<0.05.
(I) Define the domains of the CIP protein that mediate the repression of Isl1 transcription activity. MEF2C enhancer-luciferase reporter was co-transfected with indicated expression plasmids (full-length isl1, full-length or truncated CIP mutants) in HEK293T cells and luciferase activity was measured. Results were presented as relative luciferase activity in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in triplicate. *P<0.05.
(J) CIP represses myocardin- and SRF-mediated transactivation of the ANF-luciferase reporter. The ANF-luciferase reporter was co-transfected with indicated expression plasmids in HEK293T cells and luciferase activity was measured. Results were presented as relative luciferase activity in which the control was assigned a value of 1. Data represent the mean ± s.d. from at least three independent experiments in triplicate. *P<0.05.