primary antibodies against mef2d  (Boster Bio)

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: bioRxiv

Article Title: Muscle-specific MEF2D⍺2 isoform regulates mice exercise capacity, muscle ketolysis, and systemic ketone body availability in mice

doi: 10.1101/2023.05.22.540988

Figure Lengend Snippet: RT-PCR analysis of the mutually exclusive α-exons in Mef2d transcript using total RNA from the indicated fetal hind limb muscle and tissues from adult wild-type mice. (B) RT-PCR analysis of Mef2d ⍺ exons using soleus RNA from line 2. The numbers indicate PSI; data are mean ± SD; n=3. The numbers indicate the percent spliced in (PSI). Bolded numbers are significant by Student T-test. (C) RT-PCR analysis of the alternative exons in Mef2c and Mef2a transcripts using total RNA from gastrocnemius muscle from line 1. Data are mean ± SD; n=3. PSI for exon is indicated and was not significantly different between the genotypes by Student’s t-test. (D) Western blot showing MEF2D protein levels in TA (upper left) and Soleus muscles (upper right) from line 2. The bottom panels show Coomassie stained blots, from the upper panels showing total protein loaded. The numbers are mean ± SD; n=3. Student’s t-test found no differences in genotypes. (E) RT-qPCR showing relative mRNA levels Mef2d, Mef2a , and Mef2c relative to Rpl30 using total RNA in the indicated muscle groups from line 2 mice. Data are mean ± SEM; n=3. ** p <0.01 (Student’s t-test).

Article Snippet: After blocking with 5% Milk-TBST (0.1% tween) for 1 hour at room temperature, membranes were incubated overnight at 4°C with primary antibodies against MEF2D (BD, 610774), GLUT4 (Millipore, 07-1404), CD36 (R&D Systems, AF2519).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Muscle-specific MEF2D⍺2 isoform regulates mice exercise capacity, muscle ketolysis, and systemic ketone body availability in mice

doi: 10.1101/2023.05.22.540988

Figure Lengend Snippet: (A) RT-PCR analysis of the mutually exclusive α-exons and alternative β exon in Mef2d transcript using total RNA from the indicated muscle groups from Wt and Mef2d⍺2 Eko mice from line 1. The numbers indicate the percent spliced in (PSI) for indicated exon. Data are mean ± SD; n=3. Bold numbers indicate significance by Student t-test. (B) Western blot showing MEF2D protein levels in TA (upper left) and Soleus muscles (upper right) from line 1. Bottom panels show Coomassie stained blots from the upper panels showing total protein loaded. The numbers indicate the relative protein level normalized to total protein by Coomassie stained blots. Data are mean ± SD; n=3. (C) RT-qPCR showing mRNA levels Mef2d, Mef2a , and Mef2c relative to Rpl30 using total RNA in the indicated muscle groups from line 1 mice. Data are mean ± SEM; n=3. No significant changes were found between the genotypes by multiple t-test in Figs B and C.

Article Snippet: After blocking with 5% Milk-TBST (0.1% tween) for 1 hour at room temperature, membranes were incubated overnight at 4°C with primary antibodies against MEF2D (BD, 610774), GLUT4 (Millipore, 07-1404), CD36 (R&D Systems, AF2519).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Muscle-specific MEF2D⍺2 isoform regulates mice exercise capacity, muscle ketolysis, and systemic ketone body availability in mice

doi: 10.1101/2023.05.22.540988

Figure Lengend Snippet: (A) RNA-sequencing data using poly-A selected RNA from WT and MEF2D⍺2 Eko quadriceps muscles were aligned to mm10 UCSC browser showing Mef2d ⍺ exons region. (B) Graph showing number of genes that were down or upregulated in MEF2D⍺2 Eko muscles in comparison to age and sex-matched WT muscles (FDR <0.05). (C) RT-qPCR showing relative expression of Bdh1, Oxct1, and Acat1 transcripts normalized to Hprt transcript levels in WT and MEF2D⍺2 Eko soleus muscles from line 1. Data are mean ± SEM; n ≥ 6. ** p <0.01, *** p <0.001 (Student’s t-test). (D) Western blot showing BDH1 level in soleus muscles of indicated mice from line 1. The panel on the right show BDH1, OXCT1, and ACAT1 level when normalized to total protein loaded as estimated by Coomassie staining of the same blot. Data are mean ± SEM, * p <0.05, ** p <0.01, **** p <0.0001 (Student’s t-test).

Article Snippet: After blocking with 5% Milk-TBST (0.1% tween) for 1 hour at room temperature, membranes were incubated overnight at 4°C with primary antibodies against MEF2D (BD, 610774), GLUT4 (Millipore, 07-1404), CD36 (R&D Systems, AF2519).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Western Blot, Staining