mouse anti myh6  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: α- and β-myosin II can be non-uniformly distributed within the cardiac sarcomere

doi: 10.1016/j.isci.2025.112233

Figure Lengend Snippet: α- and β-myosin II can exhibit unique distributions within cardiac sarcomeres (A) Representative example of IF of endogenous β-myosin II (top) and expression of mEGFP- MYH6 (middle) in a hiCM; bottom shows overlay. Scale bars: left 10 μm, right 2 μm. (B) Line scan from yellow boxes in (A). (C) Average of all line scans (20 cells, 772 sarcomeres, 3 independent experiments). Lines show population-averaged mean value while shaded bands show ± standard error of the mean. (D) Top: expression of mNeon- MYH7 and IF of endogenous α-myosin II in a representative hiCM. Bottom: Average of all line scans (15 cells, 456 sarcomeres, 3 independent experiments). Lines show population-averaged mean value while shaded bands show ± standard error of the mean. White brackets denote individual filament stacks. Scale bar: 2 μm. (E) Distance each myosin II extended from filament center in hiCMs (β-myosin II IF: 14 cells, 280 measurements; α-myosin II IF: 15 cells, 300 measurements; mNeon- MYH7 (β expressed): 15 cells, 300 measurements; mEGFP- MYH6 (α expressed): 15 cells, 300 measurements). p -values in grey (Welch's t-test). (F) Simplified model of the potential arrangement of β-myosin II (magenta) and α-myosin II (green) in thick filaments of iPSC-derived cardiac myocyte sarcomeres. (G) Left: IF using antibodies against β-myosin II (top) and α-myosin II (bottom) in different sections of human ventricle; right: distance the fluorescent signal of each myosin extended from the filament center (β-myosin II: 280 measurements; α-myosin II: 321 measurements). Scale bar: 2 μm. p -values in grey (Welch's t-test). (H) Left: individual β-myosin II (ventricle) and α-myosin II (atrium) whole-mount immunofluorescence stains in 72 h post-fertilization zebrafish embryos; right: distance the fluorescent signal of each myosin extended from the filament center (β-myosin II: 14 embryos, 222 measurements; α-myosin II: 13 embryos, 102 measurements). White brackets in each image denote individual filament stacks. Scale bar: 2 μm. p -values in grey (Welch's t-test); (I) AlphaFold3-predicted model of α-myosin II/β-myosin II heavy chain ( MYH6 / MYH7 genes, respectively) motors + motor/lever arms, modeled with MYL2 / MYL3 (gray), which encode the ventricular essential/regulatory myosin light chains (a similar AlphaFold3-predicted model, but modeled with atrial essential/regulatory myosin light chains instead of ventricular light chains, is shown in ). N1, 2, and 3 in (E), (G), and (H) represent individual biological replicates, color-coded by replicate.

Article Snippet: MYH6: atrium-specific myosin heavy chain mouse IgG , Developmental Studies Hybridoma Bank (DSHB) , Cat# S46; RRID: AB_528376.

Techniques: Expressing, Derivative Assay, Immunofluorescence

Journal: iScience

Article Title: α- and β-myosin II can be non-uniformly distributed within the cardiac sarcomere

doi: 10.1016/j.isci.2025.112233

Figure Lengend Snippet:

Article Snippet: MYH6: atrium-specific myosin heavy chain mouse IgG , Developmental Studies Hybridoma Bank (DSHB) , Cat# S46; RRID: AB_528376.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Cell

Article Title: Multimodal charting of molecular and functional cell states via in situ electro-sequencing

doi: 10.1016/j.cell.2023.03.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-MYH6 antibody , Sigma-Aldrich , Cat#AMAB90950; RRID: AB_2665730.

Techniques: Recombinant, Microscopy, In Situ, Sequencing, Software

Journal: Clinical Epigenetics

Article Title: Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

doi: 10.1186/s13148-024-01653-7

Figure Lengend Snippet: Establishment of hESCs-derived in vitro cardiac differentiation. A Scheme of in vitro cardiac differentiation derived from hESCs. B Immunofluorescence of cTnT after 12-day differentiation (Scale bar = 20 μm). C-G The relative mRNA expression levels of cardiac markers ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT) during 12-day differentiation were tested by qRT-PCR ( n = 3-4). H The relative protein levels of cardiac markers (GATA4, NKX2.5, TBX5, MYH6 and cTnT) during 12-day differentiation were detected by western blotting ( n = 3). I Flow cytometry analysis of cTnT-positive cell during 12-day differentiation ( n = 3). D0, D3, D6, D9 and D12 indicated the time points at Day 0, Day 3, Day 6, Day 9 and Day 12, respectively. Quantitative data were presented as mean ± SEM, while statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: To block non-specific binding, the membranes were incubated in 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies including rabbit anti-α-tubulin (diluted 1:1000, Abcam, ab52866), rabbit anti-GAPDH (diluted 1:1000, Cell Signaling Technology, #5174), rabbit anti-TET2 (diluted 1:1000, Cell Signaling Technology, #45010), rabbit anti-TBX5 (diluted 1:1000, Abcam, ab137833), mouse anti-NKX2-5 (diluted 1:1000, Abcam, ab91196), rabbit anti-GATA4 (diluted 1:1000, Abcam, ab134057), rabbit anti-cTnT (diluted 1:1000, Abcam, ab209813) and mouse anti-MYH6 (diluted 1:1000, Abcam, ab50967).

Techniques: Derivative Assay, In Vitro, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Clinical Epigenetics

Article Title: Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

doi: 10.1186/s13148-024-01653-7

Figure Lengend Snippet: Inhibition of hESC-derived cardiac differentiation by miR-20b-5p. A-C qRT-PCR analysis of miR-20b-5p level during 12-day cardiac differentiation in hESCs-CMs A , hiPSCs-CMs B and mESCs-CMs C ( n = 3–4). D qRT-PCR analysis of miR-20b-5p level in embryonic and neonatal heart samples (E17.5, P2 and P9) from wild-type C57BL/6 mice ( n = 3). E qRT-PCR analysis of the relative mRNA expression level of cardiac transcriptional factors ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT ) in 12-day cardiac-differentiated hESCs with treatment of m-NC (mimic-negative control), miR-20b-5p-mimic, i-NC (inhibitor-negative control) or miR-20b-5p-inhibitor ( n = 3–5). F and G Western blot analysis G and quantification F of the relative protein levels of cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor ( n = 3). H Immunofluorescence of cTnT in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (Scale bar = 10 μm). I and J Flow cytometry analysis I and quantification J of cTnT-positive cells in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor ( n = 3). D0, D3, D6, D9 and D12 indicated the time points at Day 0, Day 3, Day 6, Day 9 and Day 12, while E17.5, P2 and P9 indicated Embryonic day 17.5, Postnatal day 2 and 9, respectively. Quantitative data were presented as mean ± SEM, while statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: To block non-specific binding, the membranes were incubated in 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies including rabbit anti-α-tubulin (diluted 1:1000, Abcam, ab52866), rabbit anti-GAPDH (diluted 1:1000, Cell Signaling Technology, #5174), rabbit anti-TET2 (diluted 1:1000, Cell Signaling Technology, #45010), rabbit anti-TBX5 (diluted 1:1000, Abcam, ab137833), mouse anti-NKX2-5 (diluted 1:1000, Abcam, ab91196), rabbit anti-GATA4 (diluted 1:1000, Abcam, ab134057), rabbit anti-cTnT (diluted 1:1000, Abcam, ab209813) and mouse anti-MYH6 (diluted 1:1000, Abcam, ab50967).

Techniques: Inhibition, Derivative Assay, Quantitative RT-PCR, Expressing, Negative Control, Western Blot, Immunofluorescence, Flow Cytometry

Journal: Clinical Epigenetics

Article Title: Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

doi: 10.1186/s13148-024-01653-7

Figure Lengend Snippet: Reversal of TET2 knockdown-mediated suppression in hESCs-derived cardiac differentiation by inhibiting miR-20b-5p. A qRT-PCR analysis of the relative mRNA expressions levels of TET2 and cardiac transcriptional factors ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT ) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC (mimic-negative control) or miR-20b-5p-mimic ( n = 3). B qRT-PCR analysis of the relative mRNA expressions levels of TET2 and cardiac transcriptional factors ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT ) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of i-NC (inhibitor-negative control) or miR-20b-5p- inhibitor ( n = 3). C and E Western blot analysis C and quantification E of the relative protein levels of TET2 and cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC or miR-20b-5p-mimic ( n = 3). D and F Western blot analysis D and quantification F of the relative protein levels of TET2 and cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of i-NC or miR-20b-5p-inhibitor ( n = 3). G-I Flow cytometry analysis G and quantification H and I of cTnT-positive cell in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor ( n = 3). J and K Immunofluorescence staining of TET2 J and cTnT K in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (Scale bar = 10 μm). Quantitative data were presented as mean ± SEM. Statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as * P < 0.05, ** P < 0.01 and *** P < 0.001, while n.s. indicated non-significance

Article Snippet: To block non-specific binding, the membranes were incubated in 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies including rabbit anti-α-tubulin (diluted 1:1000, Abcam, ab52866), rabbit anti-GAPDH (diluted 1:1000, Cell Signaling Technology, #5174), rabbit anti-TET2 (diluted 1:1000, Cell Signaling Technology, #45010), rabbit anti-TBX5 (diluted 1:1000, Abcam, ab137833), mouse anti-NKX2-5 (diluted 1:1000, Abcam, ab91196), rabbit anti-GATA4 (diluted 1:1000, Abcam, ab134057), rabbit anti-cTnT (diluted 1:1000, Abcam, ab209813) and mouse anti-MYH6 (diluted 1:1000, Abcam, ab50967).

Techniques: Derivative Assay, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Flow Cytometry, Immunofluorescence, Staining