1. Volume 24, Issue 8, Pages 1301-1310 (August 2016)
FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes 
Alisson Campos Cardoso, Ana Helena Macedo Pereira, Andre Luis Berteli Ambrosio, Silvio Roberto Consonni, Renata Rocha de Oliveira, Marcio Chain Bajgelman, Sandra Martha Gomes Dias, Kleber Gomes Franchini 
Structure 
Volume 24, Issue 8, Pages (August 2016)
DOI: /j.str
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2. Structure 2016 24, 1301-1310DOI: (10.1016/j.str.2016.06.003)
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3. Figure 1
FAK Expression, Activity and Localization in Non-stretched and Stretched NRVMs
(A) Representative pY397-FAK, FAK, MEF2, and GAPDH-specific immunoblots from extracts of non-stretched (NS) or 1-hr stretched (ST) NRVMs.
(B) Representative FAK, MEF2, GAPDH, and Sm-D1 immunoblots from cytosolic (c) and nuclear (n) extracts of NS or ST NRVMs.
(C) Confocal maximum-intensity z-projections of NS or ST NRVMs stained with anti-FAK (green) and DAPI (blue). Merged images are composed of FAK and DAPI.
(D) Confocal maximum-intensity z-projections of ST NRVMs in which the primary antibody was omitted and used as a negative control. The merge image is composed by Alexa 488 and DAPI.
The scale bar represents 20 μm. See also Figure S1. The densitometric readings of immunoblots are shown in Figure S2 and uncropped blots are shown in Figure S6A.
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4. Figure 2 FAK Interacts with MEF2 in Stretched Cardiomyocytes
(A) MEF2 immune complex isolated from cytosolic and nuclear extracts of NS and ST NRVMs. Samples were probed using specific antibodies for FAK and MEF2 as indicated.
(B) Representative co-immunoprecipitation experiment using anti-FAK, anti-MEF2, and anti-Sm-D1 antibodies and nuclear extracts from NS and ST NRVMs.
(C) Schematic representation of mouse FAK and MEF2 domains and the residue numbers of the related amino acids.
(D) Pull-down experiments using constructs of FAK domains fused to GST (bottom) and nuclear extracts of ST NRVMs. The upper panel shows the representative MEF2 immunoblot used to detect the association with the constructs of FAK domains.
(E) Fluorescence polarization curve of FAT domain binding to FITC-MEF2C_95. Kd is shown as the best-fit value of three repeat experiments, performed in three technical replicates.
See also Figure S4. The error bars are SD. The uncropped blots are shown in Figure S6B.
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5. Figure 3 Structure of the FAK FAT Domain Bound to MEF2C_95
(A) Overall views of the FAT:MEF2C_95 complex crystal structure in cartoon representation.
(B and C) FAT:MEF2C_95 complex interface showing the α2 and α3 helices of FAT (gray) and helices H2 of the MEF2 dimer (green and cyan). Interacting residues are shown in stick model mode, colored by element: carbon (by chain), nitrogen (blue), and oxygen (red). Residues that make hydrophobic interactions are shown as sticks and dots, and labeled with black fonts.
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6. Figure 4
SAXS Analysis, Mutagenesis, and Structural Comparison of the FAT:MEF2C_95 Complex with Related Structures
(A) Experimental SAXS data of the FAT:MEF2C_95 complex (blue dots). The superposition of the calculated scattering curve of the best model obtained by BUNCH and the crystallographic model are shown in red and green lines, respectively.
(B) Ab initio model, calculated from the SAXS data, shown as blue beads and the complex as a cartoon.
(C) Pull-down assays performed with GST-MEF2C_95 and FAT-WT or mutants, as indicated. Top panel: localization of the mutant residues showed as spheres. Middle panel: FAK FAT immunoblot used to detect the association with the GST-MEF2C_95 construct. Bottom panel: Coomassie-stained SDS-PAGE of purified FAT-WT or mutants. MW, molecular weight.
(D) Backbone superposition of different structures of protein:MEF2 complexes (FAK FAT, Cabin1, HDAC9, and p300). The structures are superimposed by the Cα backbone of the MEF2 chain A (green) from the four related structures. The DNA from the related structures is shown in orange.
See also Figures S3 and S4 and Table S1. The uncropped blot is shown in Figure S6C.
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7. Figure 5 FAK Activates MEF2 Transcriptional Activity
(A) Luciferase reporter assay performed in H9c2 cardiomyoblasts co-transfected with 3xMEF2-LUC and full-length myc-tagged FAK (FAK) or mutants, as indicated. The lower panel shows representative Myc-tag and GAPDH immunoblotting used as a loading control.
(B) Representative scheme of rat Jun promoter and primers used for ChIP-PCR assay (upper). The lower panel shows the representative PCR of the ChIP assay for the Jun promoter region containing a conserved MEF2-binding site (106 bp upstream from the transcription starting site). Myocardial extracts were isolated from samples of rat left ventricle after sham-operation (SO) or transverse aortic constriction (TAC; 1 hr). ChIP assays were performed with antibody to FAK, MEF2, or no antibody (No-Ab) used as control for immunoprecipitation. Input shows the PCR product of total chromatin without prior immunoprecipitation (n = 4).
(C) Real-time PCR of Jun gene in samples of NRVMs transfected with constructions as indicated.
(D) Luciferase reporter assay performed in NRVMs co-transfected with a luciferase reporter gene containing MEF2-reponsive Jun promoter (pJTXGL3-LUC) and constructs as indicated. Full-length MEF2C (M), myc-FAK (FAK), myc-FAK mutant K955E (M + K955E), empty myc vector (Vector).
Data in (A, C, and D) represent the average fold induction from four independent experiments; error bars show SEM. ∗p < 0.05 vs. CT; #p < 0.05 vs. M by one-way ANOVA followed by Bonferroni's multiple comparison test.
See also Figure S5. The uncropped blots are shown in Figure S6D.
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