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Mef2c

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MEF2C is a transcription factor that plays a role in the regulation of gene expression during muscle development and differentiation. It is involved in the control of cell growth, survival, and differentiation in various cell types. MEF2C is an important regulator of skeletal and cardiac muscle-specific genes.

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Lab products found in correlation

Mef2d, by BD (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Mef2a, by Abcam (2 mentions) Gata4, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Lsm 880, by Zeiss (2 mentions) Hdac4, by GeneTex (1 mentions) Isolectin b4 ib4, by Vector Laboratories (1 mentions) Ha tag, by Santa Cruz Biotechnology (1 mentions) Hdac4, by Santa Cruz Biotechnology (1 mentions) Stat5, by Santa Cruz Biotechnology (1 mentions) Lamin b, by Santa Cruz Biotechnology (1 mentions) β actin, by Abcam (1 mentions) Bcl 2, by Santa Cruz Biotechnology (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Ipvh00010, by Merck Group (1 mentions) Ripa buffer, by Merck Group (1 mentions) Gapdh, by Merck Group (1 mentions)

22 protocols using mef2c

1

Hindbrain, Retina, and Tumor Immunohistochemistry

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Hindbrains and retinas were processed as described (del Toro et al. 2010 (link); Pitulescu et al. 2010 (link); Fantin et al. 2013 (link)), and tumors and E10 embryos were fixed in 4% PFA for 1 h on ice. After incubation in blocking solution (10% normal donkey serum, 0.1% [v/v] Triton X-100 in PBS), samples were incubated overnight at 4°C with the designated primary antibodies (MEF2A [Abcam], MEF2B [Abcam], MEF2C [Cell Signaling], MEF2D [BD], EP300 [Active Motif], HDAC4 [GeneTex], DLL4 [R&D Systems] isolectin B4 [IB4] [Vector Laboratories], and Erg [Abcam]) in 0.1% PBS-T. Samples were washed in PBS-T and incubated for 3 h with suitable species-specific Alexa fluor- or biotin-conjugated secondary antibodies in 0.1% PBS-T. Total numbers of branch points and tip cells and retinal outgrowth length were measured after IB4 staining using ImageJ software from pooled images of retinas from at least two independent litters.
Sacilotto N., Chouliaras K.M., Nikitenko L.L., Lu Y.W., Fritzsche M., Wallace M.D., Nornes S., García-Moreno F., Payne S., Bridges E., Liu K., Biggs D., Ratnayaka I., Herbert S.P., Molnár Z., Harris A.L., Davies B., Bond G.L., Bou-Gharios G., Schwarz J.J, & De Val S. (2016). MEF2 transcription factors are key regulators of sprouting angiogenesis. Genes & Development, 30(20), 2297-2309.
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2

Western Blot Antibody Characterization Protocol

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Western blot antibodies were obtained from Cell Signaling Technology unless specified otherwise: MEF2C (catalog 5030), Cleaved NOTCH1 Val1744 (catalog 4147), MAML1 (catalog 11959), STAT5 (catalog 94205), phospho-STAT5 Tyr694 (catalog 9351), HA-tag (catalog 3724), DDK-tag (catalog 2368), HDAC4 (catalog 15164), phospho–HDAC4 Ser246/HDAC5 Ser259/HDAC7 Ser155 (catalog 3443), BCL2 (Santa Cruz Biotechnology Inc., sc-130308), LAMIN B (Santa Cruz Biotechnology Inc., sc-6216), β-actin (Abcam, ab6276), phospho-Ser222 MEF2C (PhosphoSolutions, p1208-222), and SIK3 (Sigma-Aldrich, HPA048161).
Canté-Barrett K., Meijer M.T., Cordo’ V., Hagelaar R., Yang W., Yu J., Smits W.K., Nulle M.E., Jansen J.P., Pieters R., Yang J.J., Haigh J.J., Goossens S, & Meijerink J.P. (2022). MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus. JCI Insight, 7(13), e150363.
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3

Protein Extraction and Western Blotting Protocol

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Cell samples and flash-frozen tissues were processed with RIPA buffer (MilliporeSigma, R0278). The tissue lysates were further dissociated with mechanical dissociation (Precellys Evolution), and protein concentration was quantified by BCA assay (Thermo Fisher, 23225). Regular wet transfer was performed on polyvinylidene fluoride membranes (Millipore, IPVH00010). Blocking and antibody incubation were performed in 5% milk in TBS-Tween 0.1%. The following antibodies were used: GAPDH (MilliporeSigma, MAB374), α-tubulin (MilliporeSigma, T6199), NET39 (MilliporeSigma, HPA070252), MEF2C (Cell Signaling Technology, 5030), MYOD (Santa Cruz Biotechnology Inc., sc-377460), MYOG (Santa Cruz Biotechnology Inc., sc-12732 X), vinculin (VCL) (MilliporeSigma, V9131), HRP-conjugated streptavidin (Thermo Fisher, N100), γH2A.X (MilliporeSigma, 05-636), total ATM (Cell Signaling Technology, 2873T), and phosphorylated ATM (Ser1981) (Santa Cruz Biotechnology Inc., sc-47739). For blotting of total and phosphorylated ATM as well as γH2A.X, blocking was performed in 5% BSA in TBS-Tween 0.1%.
Zhang Y., Ramirez-Martinez A., Chen K., McAnally J.R., Cai C., Durbacz M.Z., Chemello F., Wang Z., Xu L., Bassel-Duby R., Liu N, & Olson E.N. (2023). Net39 protects muscle nuclei from mechanical stress during the pathogenesis of Emery-Dreifuss muscular dystrophy. The Journal of Clinical Investigation, 133(13), e163333.
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4

Western Blot Analysis of Cardiac Markers

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Cells were homogenized in ice cold lysis buffer containing proteinase and phosphatase inhibitor cocktail. The primary antibodies utilized in this study were as follows: OCT4 (sc-5279; Santa Cruz Biotechnology), Nanog (A3233 ABclonal), NKX2.5 (ab91196; Abcam), α-actinin (6487; Cell Signaling Technology), CX43 (3512 Cell Signaling Technology), GATA4 (5851; Cell Signaling Technology), MEF2C (5030; Cell Signaling Technology), cTnI (ab47003; Abcam), p-ERK (4370; Cell Signaling Technology), ERK (5013 Cell Signaling Technology), β-Catenin (8480; Cell Signaling Technology), p-cadherin (2189; Cell Signaling Technology), p-STAT3 (RT1490; HuaBio), STAT3 (ET1605; HuaBio), and GAPDH (5174; Cell Signaling Technology). Blots were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA).
Zhu X., Ding S., Li H., Zhang Z., Xu L., Wu J., Wang X., Zou Y., Yang X, & Ge J. (2020). Disruption of histamine/H1R signaling pathway represses cardiac differentiation and maturation of human induced pluripotent stem cells. Stem Cell Research & Therapy, 11, 27.
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5

Immunostaining of Cardiomyocytes for Lineage Markers

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The cells were immunostained as described previously21 (link). Briefly, cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 (Sigma), blocked in 10% normal goat serum (Vector Laboratories), and then incubated at 4 °C overnight with primary antibodies against MESP1 (1:100, Aviva Systems Biology), MEF2C (1:100, Cell Signaling), GATA4 (1:300, Santa Cruz Biotechnology), ISL1 (1:100, Developmental Studies Hybridoma Bank), and NKX2-5 (1:200, Santa Cruz Biotechnology). Primary antibodies were detected with DyLight 549-conjugated secondary antibodies, and nuclei were counterstained with DAPI (Sigma).The immunostaining of isolated NRCMs were stained with α-actinin antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using the In situ Cell Death Detection Kit (Roche Applied Science, Germany) to detect the cell death.
Wu Q., Wang J., Tan W.L., Jiang Y., Wang S., Li Q., Yu X., Tan J., Liu S., Zhang P., Tiang Z., Chen Z., Foo R.S, & Yang H.T. (2020). Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis. Cell Death & Disease, 11(5), 354.
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6

Cardiopoietic Phenotyping of [89Zr]Zr-DBN-CPs

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[89Zr]Zr-DBN-CPs and unlabeled CPs were seeded on 6-well plates and cultured in AMEM with 5% PLT Max, 1% Glutamax, 1 unit/mL heparin, and 1% penicillin-streptomycin. After two days of culture, cells were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI) and immunostained with primary antibodies against myocyte enhancer factor 2C (MEF2C, 1:400, Cell Signaling Technologies, TX, USA), T-box transcription factor 5 (TBX5, 1:5000, Abcam, MA, USA) and mesoderm posterior bHLH transcription factor 1 (MESP1,1:250, Novus Bio, CO, USA) for cardiopoietic phenotyping [26 (link)]. The secondary antibody against MEF2C and TBX5 was conjugated with Alexa Fluor 555 (Excitation (Ex): 555 nm, Emission (Em): 590 nm), and a secondary antibody against MESP1 was conjugated with Alexa Fluor 488 (Ex: 496 nm, Em: 519 nm). Fluorescent images were acquired using either a Zeiss Axioplan epifluorescence wide field microscope (Carl Zeiss AG, Oberkochen, Germany) using a 10X non-immersion objective (numerical aperture 0.3) with an HBO mercury lamp for DAPI, Alexa Fluor 488 and Alexa Fluor 555 channels or with LSM780 Confocal microscope (40X Water; numerical aperture 1.2). Expression of MEF2C, TBX5 and MESP1 in [89Zr]Zr-DBN-CPs was quantified using Zen software (Carl Zeiss) and compared with unlabeled CPs (control) (n > 200 cells from independent experiments) [17 (link), 26 (link)].
Bansal A., Pandey M.K., Yamada S., Goyal R., Schmit N.R., Jeon R., Nesbitt J.J., Witt T.A., Singh R.D., Gunderson T.M., Boroumand S., Li M., Crespo-Diaz R.J., Hillestad M.L., Terzic A., Behfar A, & DeGrado T.R. (2020). [89Zr]Zr-DBN labeled cardiopoietic stem cells proficient for heart failure. Nuclear medicine and biology, 90-91, 23-30.
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7

Histone Deacetylase 5 and MEF2C Interaction

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Histone deacetylase 5 (HDAC5, Santa Cruz, SC-11419; 2 µg for ChIP; HDAC5, Cell Signaling, 2082; 1:50 for Co-IP); MEF2C (Cell Signaling, 5030; 1:50 for ChIP; 1:1000 for Co-IP WB; 1:500 for ICC WB); TUJ1 (Millipore, MAB1637; 1:1000 for ICC WB); MAP-2 Alexa Fluor 680 (Invitrogen, A21109; 1:3000 for Co-IP WB).
Puang S.J., Elanggovan B., Ching T, & Sng J.C. (2020). MEF2C and HDAC5 regulate Egr1 and Arc genes to increase dendritic spine density and complexity in early enriched environment. Neuronal Signaling, 4(3), NS20190147.
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8

CRISPR-Cas9 Knockout Efficiency Validation

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Synthetically modified sgRNA constructs were purchased from Synthego (refer to Supplementary Table S1 for sgRNA sequences). Ribonucleoprotein (RNP) assembly was performed by mixing 2 to 3 sgRNAs (a total of 120 pmol/L) with 8.5 μg recombinant Cas9 (Invitrogen A36499). The resulting RNP mix was electroporated into 0.3 × 106 cells using a Lonza 4D Nucleofector, program DJ-100, in 20 μL Nucleocuvette strips (Lonza V4XC-2032). Unless otherwise noted, cells were incubated in media for 72 hours postelectroporation before subsequent analyses. Knockout efficiency was confirmed by Western blotting (Supplementary Fig. S4A). The following antibodies were used: actin (Santa Cruz sc-47778), IRF8 (Cell Signaling 5628s), MEF2C (Cell Signaling 5030s), MYB (Abcam ab45150). A guide RNA targeting the AAVS1 “safe harbor” locus was used as a negative control (44 (link)).
Eagle K., Harada T., Kalfon J., Perez M.W., Heshmati Y., Ewers J., Koren J.V., Dempster J.M., Kugener G., Paralkar V.R., Lin C.Y., Dharia N.V., Stegmaier K., Orkin S.H, & Pimkin M. (2022). Transcriptional Plasticity Drives Leukemia Immune Escape. Blood Cancer Discovery, 3(5), 394-409.
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9

Antibody-Coated Magnetic Bead Assay

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Protein A- or G-coated magnetic beads were purchased from Millipore Sigma (Saint Louis, MO). The native human C1Q was purchased from Abcam (Boston, MA, USA). Papain solution was purchased from Millipore Sigma, MO. The CD14, CD34, CSTA, UBC, and BST1 antibodies were purchased from R&D Systems (Minneapolis, MN, USA), the P3H1, RGS12, and GUK1 monoclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and the MEF2C and PHACTR4 monoclonal antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA) and Abcam, respectively. The anti-human IgG antibody was purchased from Jackson ImmunoResearch (West Grove, PA).
Tang C., Fang M., Tan G., Zhang S., Yang B., Li Y., Zhang T., Saxena R., Mohan C, & Wu T. (2022). Discovery of Novel Circulating Immune Complexes in Lupus Nephritis Using Immunoproteomics. Frontiers in Immunology, 13, 850015.
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10

Western Blot Analysis of Myogenic Markers

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C2C12 mouse myoblasts or human myoblast cell HSMMs were washed with PBS and lysed in RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP 40, 0.1% SDS, 0.5% deoxycholate, Protease Inhibitor Cocktail (Roche)]. Total protein (~20 μg) was separated in SDS-PAGE, transferred to PVDF membranes and immunoblotted with specific antibodies; AKAP6 (1:3000, Covance PRB-451P), AKAP79 (1:3000, BD 610314), MEF2D (1:3000, BD 610774), MyHC (1:3000, Sigma M4276), MEF2C (1:3000, Cell signaling #9792), AKAP-Lbc (1:3000, Santa Cruz sc-9336), AKAP12 (1:3000, Santa Cruz sc-33578), MEF2A (1:3000, Santa Cruz sc-313), myogenin (1:3000, Santa Cruz sc-12732), MyoD (1:3000, Santa Cruz sc-760), actin (1:3000, Santa Cruz sc-1616), α-tubulin (1:5000, Calbiochem CP06). After incubation with horseradish peroxidase conjugated secondary antibody (1:5000, Santa Cruz sc-2005, sc-2004, sc-2020) for 1 h, immunoreactive bands were visualized with enhanced chemiluminescence with Novex® ECL Chemiluminescent Substrate Reagent (Invitrogen).
Lee S.W., Won J.Y., Yang J., Lee J., Kim S.Y., Lee E.J, & Kim H.S. (2015). AKAP6 inhibition impairs myoblast differentiation and muscle regeneration: Positive loop between AKAP6 and myogenin. Scientific Reports, 5, 16523.
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