Journal: Cytotechnology

Article Title: Novel regulations of MEF2-A, MEF2-D, and CACNA1S in the functional incompetence of adipose-derived mesenchymal stem cells by induced indoxyl sulfate in chronic kidney disease

doi: 10.1007/s10616-016-9983-0

Figure Lengend Snippet: Schematic summary of IS induced ADMSCs functionally incompetence via MEF2A, MEF2D and CACNA1S. The malfunction of ADMSCs subsequently resulted in declined the capacity of stem cells proliferation, migration and wound healing power. Meanwhile, cell apoptosis process has been progressively escalated concomitant with cells blocked at G1 phase. These features eventually hold back the regeneration capacity of chronic kidney disease patients

Article Snippet: Following the manufacturer’s instructions, the primary antibodies (polyclonal antibodies) against phospho-MEF2A (Ser408)(1:1000, Cell Signaling Technology, Danvers, MA, USA), against phosphor-MEF2D (Ser444)(1:1000, GeneTex, Irvine, CA, USA), against CACNA1S (1:1000, GeneTex), and against actin (1:1000, GeneTex) were first incubated with the antigen at a 1 μg: 1 μg ratio at room temperature for 2 h as a pre-absorption control.

Techniques: Migration

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 5. Dex reversed H/R-induced upre gulation of miR-665 and downregulation of MEF2D. (A-B) Expressions of miR-665 and MEF2D mRNA were examined by qRT-PCR. (C-D) Expressions of MEF2D protein were detected by Western blot. Quantitative analyses of protein band intensity. GAPDH served as an internal control for sample loading. (E) Predicted duplex formation between MEF2D 3′- UTR and miR-665. (F) Dual luciferase gene reporter assay manifested that miR- 665 could directly bind with MEF2D. (n = 3 per group). **p < 0.01, ***p < 0.001, ****p < 0.0001. qRT- PCR, quantitative reverse transcription PCR; UTR, untranslated region; MEF2D, myocyte enhancer factor 2D.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Quantitative RT-PCR, Western Blot, Control, Luciferase, Reporter Assay, Reverse Transcription

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 6. Dex improved cell viability and decreased apoptosis of H9c2 cells undergoing H/R via downregulation of miR-665 followed by upregulation of MEF2D. The H9c2 cells were transfected with miR-665mimics or co-transfection of miR-665mimics with pcDNA-MEF2D for 48 h before treatment with 10 nM Dex for 1 h. (A) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on morphology of H9c2 cells in each group were observed using an inverted microscope (scale bars, 100 µm). (B) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on the cell viability were detected using CCK-8 assay. (C-F) The effects of miR-665 overexpression or miR-665 and MEF2D simultaneous overexpression on the cell apoptosis were gauged by flow cytometry. The apoptotic rates were presented as addition of the percentages of cells at early apoptotic phase and late apoptotic phase. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Transfection, Cotransfection, Over Expression, Inverted Microscopy, CCK-8 Assay, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 7. Dex protected against pyroptosis of H9c2 cells undergoing H/R via downregulation of miR-665 followed by upregulation of MEF2D. (A) Protein bands of MEF2D, IL-1β, IL-18, NLRP3, ASC, C-Caspase-1, and GSDMD were evaluated by Western blot. (B-C) Expressions of miR-665 and MEF2D mRNA were detected by qRT- PCR. (D-J) Quantitative analyses of protein band intensities of MEF2D, IL-1β, IL-18, NLRP3, ASC, C-Caspase-1, and GSDMD. GAPDH served as an internal control for sample loading. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis.

doi: 10.1016/j.biopha.2023.115255

Figure Lengend Snippet: Fig. 8. Dex facilitated nuclear translocation of Nrf2 regulated by MEF2D in H/R-treated H9c2 cells. (A) Expressions of cytoplasmic Nrf2 and nuclear Nrf2 proteins were determined by Western blot in H/R-treated H9c2 cells subjected to Dex pretreatment. GAPDH and Lamin B were used as internal references for sample loading respectively. (B-C) Quantitative analyses of the expression levels of cytoplasmic Nrf2 and nuclear Nrf2. (D) Expressions of cytoplasmic Nrf2 and nuclear Nrf2 proteins were detected by Western blot in H/R-treated H9c2 cells undergoing Dex pretreatment and transfection of miR-665mimics or co-transfection of miR-665mimics with pcDNA-MEF2D respectively. GAPDH and Histone3 acted as internal references for sample loading respectively. (E-F) Quantitative analyses of the expression levels of cytoplasmic Nrf2 and nuclear Nrf2. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant. Nrf2, nuclear factor erythroid 2-related factor 2.

Article Snippet: The proteins, which were subjected to 10% SDS-PAGE gels, were transferred onto PVDF membranes and probed using primary antibodies against MEF2D (1:1000, Affinity, AF7888), Nrf2(1:2000, Affinity, AF0639), NLRP3 (1:1000, Abcam, Ab263899), ASC (1:1000, Affinity, DF6304), cleavedcaspase-1 (1:1000, Affinity, AF4005), GSDMD (1:1000, Abclonal, A20197), IL-1β (1:1000, Genetex, GTX130021), IL-18(1:1000, Affinity, DF6252), Lamin B(1:1000, Boster, BA1228) and GAPDH (1:1000, GOODHERE, AB-P-R 001) as the loading control overnight at 4 ◦C.

Techniques: Translocation Assay, Western Blot, Expressing, Transfection, Cotransfection

Journal: Cell Death & Disease

Article Title: Long non-coding RNA Irm enhances myogenic differentiation by interacting with MEF2D

doi: 10.1038/s41419-019-1399-2

Figure Lengend Snippet: a RNA-FISH for detecting Irm and GAPDH in undifferentiated and differentiated C2C12 cells. Red: Irm or GAPDH . Blue: DAPI staining. Scale bar, 50 μm. b Relative abundance of Irm in total and nuclear RNAs of differentiating C2C12 cells, as detected by qRT-PCR. U6 , Xist , and GAPDH were used as endogenous controls. c A schematic representation of RNA pull-down assay. d Western blotting assay for the specific interaction of Irm with MEF2D. e A schematic representation of RNA immunoprecipitation (RIP) assay. f RIP assay showed the association of MEF2D with Irm in vivo, as detected by RT-PCR and qRT-PCR. g Deletion mapping of the MEF2D-binding region(s) in Irm . Top: western blotting for MEF2D in protein samples pulled down by the different truncated Irm constructs. Bottom: diagrams of the full-length and truncated Irms . nt: nucleotide. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; *** P < 0.05 by Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; qRT-PCR, quantitative real-time polymerase chain reaction; RNA-FISH, RNA fluorescence in situ hybridization

Article Snippet: The antibody against MEF2D (Santa Cruz Biotechnology, USA) was used for RIP.

Techniques: Staining, Quantitative RT-PCR, Pull Down Assay, Western Blot, RNA Immunoprecipitation, In Vivo, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Construct, Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization

Journal: Cell Death & Disease

Article Title: Long non-coding RNA Irm enhances myogenic differentiation by interacting with MEF2D

doi: 10.1038/s41419-019-1399-2

Figure Lengend Snippet: a The effect of Irm knockdown on the expression of myogenin and miR-206 depended on MEF2D, which was detected by qRT-PCR. b The effect of Irm knockdown on the differentiation of C2C12 cells depended on MEF2D. Fusion index was calculated. Scale bars, 50 mm. c C2C12 cells were transfected with si-Irm or si-scramble, and the luciferase reporter plasmids were generated by inserting the promoter region of myogenin or miR-206. The luciferase activities were measured 48 h after differentiation. d C2C12 cells were transfected with pc-Irm or pc-Ctrl, and the luciferase reporter plasmids were generated by inserting the promoter region of myogenin or miR-206. The luciferase activities were measured 48 h after differentiation. e Knockdown of Irm impaired the binding ability of MEF2D to myogenin and miR-206 promoters, which was determined by ChIP and qRT-PCR assays. f Knockdown of Irm impaired the binding ability of MyoD to myogenin and miR-206 promoters, which was determined by ChIP and qRT-PCR assays. g Chromatin isolation by RNA purification (ChIRP) assay was performed using even and odd antisense oligos tiling Irm and, a significant amount of genomic DNAs corresponding to myogenin and miR-206 promoters but not in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) locus was retrieved. LacZ ChIRP retrieved no signal. h Model for Irm -regulating myogenesis. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; * P < 0.05, ** P < 0.05, and *** P < 0.05 by Student’s t test. ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction

Article Snippet: The antibody against MEF2D (Santa Cruz Biotechnology, USA) was used for RIP.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Generated, Binding Assay, Isolation, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Recruitment of active calcineurin to mAKAPβ signalosomes in differentiating C2C12 skeletal myoblasts. A, mAKAPβ in striated myocyte is identical to aa residues 245–2314 of the neuronal mAKAPα (14). The three spectrin repeats (SR) required for nuclear envelope targeting are indicated (40). Binding sites are shown for those mAKAP binding partners for which there is evidence of direct binding: 3-phosphoinositide-dependent kinase-1 (PDK1) (14); adenylyl cyclase 5 (AC5) (41); MEF2 (11); phospholipase Cϵ (PLCϵ) (21); nesprin-1α (23); ryanodine receptor (RyR) (42); CaN (13); phosphodiesterase 4D3 (PDE4D3) (38); p90 ribosomal S6 kinase 3 (RSK3) (43); protein kinase A (PKA) (40); protein phosphatase 2A (PP2A) (44). HDAC5 binding to mAKAPβ has been mapped to the MEF2D site (12). MEF2D (aa 301–500) and CaN (aa 1285–1345) binding domain peptides are designated MBD and CBD, respectively. B, C2C12 cells transfected with an expression plasmid for mCherry- and FLAG-tagged CaN were cultured in GM or DM in the absence or presence of cyclosporin A (500 nm) for 3 h before immunoprecipitation using FLAG antibodies and detection of associated mAKAPβ. p (one-way ANOVA) < 0.0001. C, same as in B except using cells co-expressing CBD-mCherry or mCherry control. Note that CaN-mCherry-FLAG and the smaller CBD-mCherry (37 kDa) and mCherry (29 kDa) were readily separated by SDS-PAGE. p (two-way ANOVA for both factors and interaction) < 0.02. D, C2C12 cells cultured as in B were fractionated into cytosolic and nuclear fractions. GAPDH and lamin A antibodies were used to show the efficiency of fractionation. Detection of endogenous CaNAβ in each fraction was determined by Western blotting. p (two-way ANOVA for culture conditions and interaction) < 0.01. E, C2C12 cells were transfected with mAKAP or control siRNA before fractionation and analysis as in D. F, C2C12 cells were transfected with mCherry or CBD-mCherry expression plasmids before fractionation and analysis as in D. p (two-way ANOVA for both factors and interaction) < 0.02 for both cytosolic and nuclear fractions for E and F. n = 3 independent experiments for all panels. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Immunoprecipitation, SDS Page, Fractionation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Regulation of MEF2D Ser-444 phosphorylation. A, C2C12 cells transfected with an expression plasmid for GFP- and FLAG-tagged MEF2D were cultured in GM or DM in the absence or presence of cyclosporin A (500 nm) for 3 h before immunoprecipitation using FLAG antibodies and Western blotting using MEF2D and phospho-Ser-444 (P-MEF2D) antibodies. p (one-way ANOVA) = 0.0002. B–D, same as in A except using cells co-transfected with mAKAP or control siRNA (B), mCherry or CBD-mCherry expression plasmids (C), or mCherry or MBD-mCherry expression plasmids. n = 3 independent experiments for all panels. p (two-way ANOVA for both factors and interaction) < 0.03 for B–D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Transfection, Expressing, Plasmid Preparation, Cell Culture, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Regulation of MEF2D sumoylation. A, C2C12 cells co-transfected with expression plasmids for GFP- and FLAG-tagged MEF2D and EYFP-tagged SUMO1 were cultured in GM or DM for 3 h before immunoprecipitation using FLAG antibodies and Western blotting using MEF2D and SUMO1 antibodies. Note that sumoylated MEF2D was not detectable in total extracts (not shown). To prevent desumoylation, 25 mm N-ethylmaleimide was added to the lysis buffer. B, HEK293 cells were co-transfected with GFP- and FLAG-tagged MEF2D mutants and EYFP-tagged SUMO1 before immunoprecipitation using FLAG antibodies and Western blotting as in A. p (one-way ANOVA) = 0.004. C, same as A except C2C12 cells transfected with mAKAP or control siRNA. p (two-way ANOVA for both factors and interaction) < 0.03. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. p (two-way ANOVA for mCherry proteins expressed and interaction) < 0.04. n = 3 independent experiments for all panels. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Regulation of MEF2D acetylation. A, C2C12 cells transfected with expression plasmids for GFP- and FLAG-tagged MEF2D were cultured in GM or DM containing 5 μm trichostatin A for 3 h before immunoprecipitation using FLAG antibodies and Western blotting using MEF2D and acetylated lysine (Ac-K) antibodies. B, HEK293 cells were transfected with GFP- and FLAG-tagged MEF2D mutants before immunoprecipitation using FLAG antibodies and Western blotting as in A. p (one-way ANOVA) = 0.008. C, same as A except using C2C12 cells transfected with mAKAP or control siRNA. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. n = 3 independent experiments for all panels. p (two-way ANOVA for both factors and interaction) < 0.04 for C and D. *, p ≤ 0.05; **, p ≤ 0.01. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Regulation of MEF2D-HDAC5 binding. A, C2C12 cells co-transfected with expression plasmids for GFP- and FLAG-tagged MEF2D and GFP-tagged HDAC5 were cultured in GM or DM for 3 h before immunoprecipitation using FLAG antibodies and Western blotting using MEF2D and HDAC5 antibodies. p (one-way ANOVA) = 0.003. B, HEK293 cells were transfected with GFP- and FLAG-tagged MEF2D mutants and GFP-HDAC5 before immunoprecipitation using FLAG antibodies and Western blotting as in A. p (one-way ANOVA) < 0.0001. C, same as A except using C2C12 cells transfected with mAKAP or control siRNA. p (two-way ANOVA for both factors and interaction) < 0.04. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. p (two-way ANOVA for growth media and interaction) < 0.04. n = 3 independent experiments for all panels. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Binding Assay, Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Regulation of MEF2D–p300 binding. A, C2C12 cells co-transfected with expression plasmids for GFP- and FLAG-tagged MEF2D and p300 were cultured in GM or DM for 3 h before immunoprecipitation using FLAG antibodies and Western blotting using MEF2D and p300 antibodies. p (one-way ANOVA) = 0.003. B, HEK293 cells were transfected with GFP- and FLAG-tagged MEF2D mutants and GFP-HDAC5 before immunoprecipitation using FLAG antibodies and Western blotting as in A. p (one-way ANOVA) = 0.02. C, same as A except C2C12 cells transfected with mAKAP or control siRNA. p (two-way ANOVA for both factors and interaction) < 0.04. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. p (two-way ANOVA for growth media and interaction) < 0.05. n = 3 independent experiments for all panels. *, p ≤ 0.05; **, p ≤ 0.01. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Binding Assay, Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Calcineurin-dependent regulation of MEF2D–p300 DNA complexes. A, C2C12 cells expressing GFP- and FLAG-tagged MEF2D were cultured in GM or DM in the absence or presence of cyclosporin A (500 nm) for 3 h before pulldown assay using a biotinylated oligonucleotide based upon a Myh7 MyoD cis-active element (24) and Western blotting using MEF2D antibodies. Myh7m is a mutant oligonucleotide control. p (one-way ANOVA) = 0.004. B, same as in A except using HEK293 cells co-expressing mutant MEF2D proteins. p (two-way ANOVA for MEF2D mutants and interaction) < 0.04. C, same as A except C2C12 cells transfected with mAKAP or control siRNA. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. p (two-way ANOVA for both factors and interaction) < 0.02 for C and D. n = 3 independent experiments for all panels. *, p ≤ 0.05; **, p ≤ 0.01. Error bars, S.E.

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

doi: 10.1074/jbc.RA118.005465

Figure Lengend Snippet: Regulation of myocyte phenotype by MEF2D Ser-444 phosphorylation. A, C2C12 cells transfected with mAKAP or control siRNA were cultured for 24 h in GM and DM before analysis by Western blotting. p (two-way ANOVA for both factors and interaction) < 0.03 for both genes. B, same as in A except with cells expressing GFP- and FLAG-tagged MEF2D WT and mutant proteins. p (two-way ANOVA for MEF2D mutants and interaction) < 0.01 for myosin; p (two-way ANOVA for MEF2D mutants) = 0.02 for myogenin. C, neonatal rat ventricular myocytes were transfected with plasmids expressing FLAG-tagged MEF2D mutants and marker GFP and stained with α-actinin (red) and FLAG (blue) antibodies. The GFP channel is shown also in grayscale below. Bar, 10 μm. Myocyte cross-section areas measured using GFP images are shown. n = 3 independent experiments for A–C. p (two-way ANOVA for both factors) < 0.05. D, model for regulation of MEF2D chromatin complexes by perinuclear mAKAPβ signalosomes. We propose that MEF2D dynamically associates with chromatin, such that transient association with mAKAPβ facilitates its regulation by CaN. HDAC nuclear export is also promoted by mAKAPβ-dependent signaling (12).

Article Snippet: An expression plasmid for FLAG- and GFP-tagged MEF2D (pEGFPN1-FLAG-MEF2D) was constructed by inserting a PCR-amplified cDNA for mouse MEF2D α1β isoform (GenBank TM number {"type":"entrez-nucleotide","attrs":{"text":"S68893","term_id":"545519","term_text":"S68893"}} S68893 ) with a C-terminal FLAG tag into the HindIII and AgeI sites of pEGFPN1 (Clontech).

Techniques: Transfection, Cell Culture, Western Blot, Expressing, Mutagenesis, Marker, Staining