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rabbit polyclonal anti myo18a antibody  (Proteintech)


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    Proteintech rabbit polyclonal anti myo18a antibody
    Homozygous <t>Myo18a</t> deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.
    Rabbit Polyclonal Anti Myo18a Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+polyclonal+anti+myo18a+antibody/MYO18A+Antibody/pmc06509511-434-0-5
    Average 94 stars, based on 16 article reviews
    rabbit polyclonal anti myo18a antibody - by Bioz Stars, 2026-10
    94/100 stars

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    1) Product Images from "A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart"

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.004560

    Homozygous Myo18a deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.
    Figure Legend Snippet: Homozygous Myo18a deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.

    Techniques Used: Knock-Out, Produced, Southern Blot, Derivative Assay

    X-gal staining and histology of Myo18atm1b embryos. A, conversion of the tm1a (knockout first) allele to the tm1b (reporter knockout) allele by crossing heterozygous knockout first (Myo18a+/tm1a) mice with ACTB-Cre mice. B, timing of embryonic lethality by timed intercrossings of heterozygous knockout reporter (Myo18a+/tm1b) mice. No viable homozygous embryos were detected after E12.5. *, data from crossing Myo18a+/tm1a × Myo18a+/tm1a. C, whole-mount X-gal staining of HET Myo18a reporter knockout embryos at E11.5 and E10.5. Scale bars, 1 mm. D, histological sections of the heart obtained from X-gal–stained WT and HOM Myo18a reporter knockout E10.5 embryos. Arrows, X-gal staining of myocardial cells. Scale bars, 250 μm. AVC, atrioventricular cushions; V, ventricle; A, atrium. E, hematoxylin and eosin (HE) staining of sagittal sections of E10.5 HOM knockout (Myo18atm1b/tm1b) and WT embryos. OFT, outflow tract; AVC, atrioventricular cushions; V, ventricle; A, atrium.
    Figure Legend Snippet: X-gal staining and histology of Myo18atm1b embryos. A, conversion of the tm1a (knockout first) allele to the tm1b (reporter knockout) allele by crossing heterozygous knockout first (Myo18a+/tm1a) mice with ACTB-Cre mice. B, timing of embryonic lethality by timed intercrossings of heterozygous knockout reporter (Myo18a+/tm1b) mice. No viable homozygous embryos were detected after E12.5. *, data from crossing Myo18a+/tm1a × Myo18a+/tm1a. C, whole-mount X-gal staining of HET Myo18a reporter knockout embryos at E11.5 and E10.5. Scale bars, 1 mm. D, histological sections of the heart obtained from X-gal–stained WT and HOM Myo18a reporter knockout E10.5 embryos. Arrows, X-gal staining of myocardial cells. Scale bars, 250 μm. AVC, atrioventricular cushions; V, ventricle; A, atrium. E, hematoxylin and eosin (HE) staining of sagittal sections of E10.5 HOM knockout (Myo18atm1b/tm1b) and WT embryos. OFT, outflow tract; AVC, atrioventricular cushions; V, ventricle; A, atrium.

    Techniques Used: Staining, Knock-Out

    Mouse ventricular myocytes express a novel isoform of Myo18A and cardiac-restricted Myo18a deletion is lethal. A, generation of cardiac-restricted Myo18a knockout mice. Mice harboring the floxed Myo18a allele (Myo18atm1c) were crossed with Tnnt2-Cre mice, in which Cre recombinase is expressed in cardiac myocytes. Conditional knockout mice were further crossed with Cre reporter (R26R-EYFP) mice to derive triple mutant (cardiac-restricted Myo18a knockout with Cre reporter) mice. B, bright field and fluorescence images of ventricular myocytes isolated from a Myo18a+/tm1c/Tnnt2-Cre/R26R-EYFP mouse. Green (EYFP) fluorescence is an indicator of Cre recombinase activity. Scale bar, 10 μm. C, no homozygous cardiac-restricted Myo18a knockout (Myo18atm1c/tm1c/Tnnt2-Cre) mice were obtained after crossing Myo18atm1c/tm1c and Myo18a+/tm1c/Tnnt2-Cre mice, indicating that conditional deletion of Myo18a in cardiac myocytes is lethal. No Myo18atm1c/tm1c/Tnnt2-Cre embryos were detected at E13.5, and three dead embryos were detected at E14.5. D, photographs of HET and homozygous cardiac-restricted Myo18a knockout embryos at day E14.5. Scale bar, 0.5 mm. E, Western blotting analyses. A custom antibody against the last 18 amino acids of mouse Myo18A (anti-Myo18A-C-term) recognizes the Myo18A isoforms Myo18Aα and Myo18Aβ. Lysates were prepared from HeLa cells transiently expressing EGFP-Myo18Aα (mouse) or EGFP-Myo18Aβ (mouse). The custom anti-Myo18A-C-term antibody did not detect Myo18A in mouse heart lysate, whereas an antibody against the coiled-coil (CC) domain of Myo18A detected a larger than expected band on the same membrane.
    Figure Legend Snippet: Mouse ventricular myocytes express a novel isoform of Myo18A and cardiac-restricted Myo18a deletion is lethal. A, generation of cardiac-restricted Myo18a knockout mice. Mice harboring the floxed Myo18a allele (Myo18atm1c) were crossed with Tnnt2-Cre mice, in which Cre recombinase is expressed in cardiac myocytes. Conditional knockout mice were further crossed with Cre reporter (R26R-EYFP) mice to derive triple mutant (cardiac-restricted Myo18a knockout with Cre reporter) mice. B, bright field and fluorescence images of ventricular myocytes isolated from a Myo18a+/tm1c/Tnnt2-Cre/R26R-EYFP mouse. Green (EYFP) fluorescence is an indicator of Cre recombinase activity. Scale bar, 10 μm. C, no homozygous cardiac-restricted Myo18a knockout (Myo18atm1c/tm1c/Tnnt2-Cre) mice were obtained after crossing Myo18atm1c/tm1c and Myo18a+/tm1c/Tnnt2-Cre mice, indicating that conditional deletion of Myo18a in cardiac myocytes is lethal. No Myo18atm1c/tm1c/Tnnt2-Cre embryos were detected at E13.5, and three dead embryos were detected at E14.5. D, photographs of HET and homozygous cardiac-restricted Myo18a knockout embryos at day E14.5. Scale bar, 0.5 mm. E, Western blotting analyses. A custom antibody against the last 18 amino acids of mouse Myo18A (anti-Myo18A-C-term) recognizes the Myo18A isoforms Myo18Aα and Myo18Aβ. Lysates were prepared from HeLa cells transiently expressing EGFP-Myo18Aα (mouse) or EGFP-Myo18Aβ (mouse). The custom anti-Myo18A-C-term antibody did not detect Myo18A in mouse heart lysate, whereas an antibody against the coiled-coil (CC) domain of Myo18A detected a larger than expected band on the same membrane.

    Techniques Used: Knock-Out, Mutagenesis, Fluorescence, Isolation, Activity Assay, Western Blot, Expressing, Membrane

    Cloning and sequencing of the novel isoform of Myo18A expressed in mouse ventricular myocytes. A, representative Sashimi plot of data obtained by mouse ventricular myocyte RNA-Seq (n = 6 mice). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles (with dashed lines) indicate exons that are abundant in the ventricular myocyte transcript but absent in the aligned annotated genomic references corresponding to transcripts for Myo18Aα and Myo18Aβ. B, domain structure of the novel cardiac isoform of Myo18A, Myo18Aγ, as well as, for comparison, the domain structures of Myo18Aα and Myo18Aβ. The recognition sites of various anti-Myo18A antibodies used in this study are indicated below. Notably, the custom (anti-Myo18A-C-term) antibody is unable to recognize the novel C terminus of Myo18Aγ. PPII, polyproline II helix; KE, KE-rich region; PDZ, PDZ domain; IQ, IQ motif. C, alignment of the N-terminal protein sequence of Myo18Aγ, derived from cloning and sequencing cDNA from mouse ventricular myocytes, with a mouse protein sequence of 219 residues from the Ensembl database (MGP_FVBNJ_P0030691).
    Figure Legend Snippet: Cloning and sequencing of the novel isoform of Myo18A expressed in mouse ventricular myocytes. A, representative Sashimi plot of data obtained by mouse ventricular myocyte RNA-Seq (n = 6 mice). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles (with dashed lines) indicate exons that are abundant in the ventricular myocyte transcript but absent in the aligned annotated genomic references corresponding to transcripts for Myo18Aα and Myo18Aβ. B, domain structure of the novel cardiac isoform of Myo18A, Myo18Aγ, as well as, for comparison, the domain structures of Myo18Aα and Myo18Aβ. The recognition sites of various anti-Myo18A antibodies used in this study are indicated below. Notably, the custom (anti-Myo18A-C-term) antibody is unable to recognize the novel C terminus of Myo18Aγ. PPII, polyproline II helix; KE, KE-rich region; PDZ, PDZ domain; IQ, IQ motif. C, alignment of the N-terminal protein sequence of Myo18Aγ, derived from cloning and sequencing cDNA from mouse ventricular myocytes, with a mouse protein sequence of 219 residues from the Ensembl database (MGP_FVBNJ_P0030691).

    Techniques Used: Cloning, Sequencing, RNA Sequencing, Comparison, Derivative Assay

    Human homolog of mouse Myo18Aγ and mouse–human sequence alignment. A, representative Sashimi plot of data obtained by RNA-Seq analysis of human atrial tissue (n = 10 subjects). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles indicate exons that are detected in the human atrial tissue transcript but absent in the aligned genomic references corresponding to transcripts (a–f) for human MYO18A. The accession numbers are as follows: MYO18Aa (NM_078471), MYO18Ab (NM_203318), MYO18Ac (NM_001346765), MYO18Ad (NM_001346766), MYO18Ae (NM_001346767), and MYO18Af (NM_001346768). B, sequence alignment of the N and C termini (N- and C-term) of mouse (Mus musculus (Mus-mus)) Myo18Aγ with human (Homo sapiens (Hom-sap)) atrial MYO18A. Identical residues are highlighted in gray and indicated by an asterisk below the alignment. Conserved mutations (residues replaced by another amino acid with similar biochemical properties) are indicated by a colon, and semiconserved mutations are denoted by a period. The red numbers below the sequences for Homo sapiens correspond to the (red) numbered exons depicted in the Sashimi plot above.
    Figure Legend Snippet: Human homolog of mouse Myo18Aγ and mouse–human sequence alignment. A, representative Sashimi plot of data obtained by RNA-Seq analysis of human atrial tissue (n = 10 subjects). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles indicate exons that are detected in the human atrial tissue transcript but absent in the aligned genomic references corresponding to transcripts (a–f) for human MYO18A. The accession numbers are as follows: MYO18Aa (NM_078471), MYO18Ab (NM_203318), MYO18Ac (NM_001346765), MYO18Ad (NM_001346766), MYO18Ae (NM_001346767), and MYO18Af (NM_001346768). B, sequence alignment of the N and C termini (N- and C-term) of mouse (Mus musculus (Mus-mus)) Myo18Aγ with human (Homo sapiens (Hom-sap)) atrial MYO18A. Identical residues are highlighted in gray and indicated by an asterisk below the alignment. Conserved mutations (residues replaced by another amino acid with similar biochemical properties) are indicated by a colon, and semiconserved mutations are denoted by a period. The red numbers below the sequences for Homo sapiens correspond to the (red) numbered exons depicted in the Sashimi plot above.

    Techniques Used: Sequencing, RNA Sequencing

    Subcellular localization of Myo18Aγ in neonatal rat ventricular cardiomyocytes. A, NRVMs were lipofected with expression plasmids encoding EGFP-tagged mouse Myo18Aγ (Myo18Aγ-EGFP; middle row) and mScarlet-I-tagged mouse Cypher 1c (middle column) or mScarlet-I–tagged rat Myl2 (right column). Alternatively, cells fixed 4 days post-transfection were labeled with antibodies directed against sarcomeric α-actinin (left column). Images were obtained by laser-scanning confocal microscopy. Scale bars, 10 μm. B, enlarged views of the rectangular regions of interest shown in the top row of A. A horizontal line was drawn across these regions of interest to obtain the corresponding density plots, shown below. a.u., arbitrary units.
    Figure Legend Snippet: Subcellular localization of Myo18Aγ in neonatal rat ventricular cardiomyocytes. A, NRVMs were lipofected with expression plasmids encoding EGFP-tagged mouse Myo18Aγ (Myo18Aγ-EGFP; middle row) and mScarlet-I-tagged mouse Cypher 1c (middle column) or mScarlet-I–tagged rat Myl2 (right column). Alternatively, cells fixed 4 days post-transfection were labeled with antibodies directed against sarcomeric α-actinin (left column). Images were obtained by laser-scanning confocal microscopy. Scale bars, 10 μm. B, enlarged views of the rectangular regions of interest shown in the top row of A. A horizontal line was drawn across these regions of interest to obtain the corresponding density plots, shown below. a.u., arbitrary units.

    Techniques Used: Expressing, Transfection, Labeling, Confocal Microscopy

    Ultrastructural analysis of Myo18a-deficient E10.5 embryo hearts. A and B, high-magnification images, obtained by transmission EM, showing well-defined sarcomeres in WT and HET Myo18a knockout (Myo18a+/tm1b) E10.5 embryo hearts. The insets indicate parallel bundled filaments. C, highly disorganized sarcomeres in HOM Myo18a knockout (Myo18atm1b/tm1b) E10.5 embryo hearts. Z, Z-disc; I, I-band; A, A-band. Scale bars, 250 nm.
    Figure Legend Snippet: Ultrastructural analysis of Myo18a-deficient E10.5 embryo hearts. A and B, high-magnification images, obtained by transmission EM, showing well-defined sarcomeres in WT and HET Myo18a knockout (Myo18a+/tm1b) E10.5 embryo hearts. The insets indicate parallel bundled filaments. C, highly disorganized sarcomeres in HOM Myo18a knockout (Myo18atm1b/tm1b) E10.5 embryo hearts. Z, Z-disc; I, I-band; A, A-band. Scale bars, 250 nm.

    Techniques Used: Transmission Assay, Knock-Out

    Myo18a and Myo18b are the predominantly expressed unconventional myosins in mouse ventricular myocytes, and Myo18Aγ is detected in both mouse cardiac and skeletal muscle. A, expression levels of unconventional myosins in mouse ventricular myocytes. Data were obtained by RNA sequence analysis (n = 6 mice), and expression levels are indexed as FPKM (fragments per kilobase of transcript per million mapped reads). B, expression levels of conventional (class II) myosins in mouse ventricular myocytes. Note that Myh6, the dominant cardiac-specific myosin heavy chain isoform in adult mice, is highly expressed (2 of the 6 samples exceeded the detection range; not shown). C, Western blot analysis. Anti-Myo18A coiled-coil domain antibodies were used to detect Myo18A isoforms expressed in HeLa cells, mouse heart, and mouse skeletal (gastrocnemius) muscle. Error bars, S.E.
    Figure Legend Snippet: Myo18a and Myo18b are the predominantly expressed unconventional myosins in mouse ventricular myocytes, and Myo18Aγ is detected in both mouse cardiac and skeletal muscle. A, expression levels of unconventional myosins in mouse ventricular myocytes. Data were obtained by RNA sequence analysis (n = 6 mice), and expression levels are indexed as FPKM (fragments per kilobase of transcript per million mapped reads). B, expression levels of conventional (class II) myosins in mouse ventricular myocytes. Note that Myh6, the dominant cardiac-specific myosin heavy chain isoform in adult mice, is highly expressed (2 of the 6 samples exceeded the detection range; not shown). C, Western blot analysis. Anti-Myo18A coiled-coil domain antibodies were used to detect Myo18A isoforms expressed in HeLa cells, mouse heart, and mouse skeletal (gastrocnemius) muscle. Error bars, S.E.

    Techniques Used: Expressing, Sequencing, Western Blot

    Generation of myeloid-restricted Myo18a knockout mice and expression of the splice variants Myo18Aα and Myo18Aβ in macrophages. A, triple mutant Myo18a cKO mice. Shown is a schematic diagram of the floxed Myo18a allele (Myo18atm1c) and conversion to a myeloid-restricted Myo18a knockout allele with a fluorescent reporter (R26R-EYFP) of Cre recombinase activity. The bright field and fluorescence images on the right, obtained by spinning disk confocal microscopy, show resident peritoneal macrophages isolated from a Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mouse. Images are 120 × 120 μm. B, Western blot analysis of purified WT (F4/80+ cells) versus conditional Myo18a knockout macrophages (EYFP+ cells). In each lane, lysate from 2 × 105 cells was loaded. C, Western blot analysis of Myo18A protein detected in the following cell lysates: untransfected (human) HeLa cells, HeLa cells transfected with mouse EGFP-Myo18Aβ1, HeLa cells transfected with mouse EGFP-Myo18Aβ2, 1.5 × 105 purified peritoneal F4/80+ cells (macrophages) from WT mice, 1.5 × 105 purified peritoneal EYFP+ (Cre recombinase–positive) cells from Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mice, and (mouse) NIH-3T3 cells. A separate blot shows the relative intensities of Myo18A alternative splicing isoforms. D, relative protein levels of Myo18Aα (α) and Myo18Aβ (β) in purified mouse resident macrophages. Error bars, S.E. E, migration plots of WT and Myo18a-deficient macrophages in a complement C5a gradient. F, summary plots of chemotactic efficiency (chemotaxis index) and mean velocity (n = 100 cells (4 mice) in each group); n.s. = not significant at the 0.05 level of significance (Mann–Whitney U test). Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blotting analyses.
    Figure Legend Snippet: Generation of myeloid-restricted Myo18a knockout mice and expression of the splice variants Myo18Aα and Myo18Aβ in macrophages. A, triple mutant Myo18a cKO mice. Shown is a schematic diagram of the floxed Myo18a allele (Myo18atm1c) and conversion to a myeloid-restricted Myo18a knockout allele with a fluorescent reporter (R26R-EYFP) of Cre recombinase activity. The bright field and fluorescence images on the right, obtained by spinning disk confocal microscopy, show resident peritoneal macrophages isolated from a Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mouse. Images are 120 × 120 μm. B, Western blot analysis of purified WT (F4/80+ cells) versus conditional Myo18a knockout macrophages (EYFP+ cells). In each lane, lysate from 2 × 105 cells was loaded. C, Western blot analysis of Myo18A protein detected in the following cell lysates: untransfected (human) HeLa cells, HeLa cells transfected with mouse EGFP-Myo18Aβ1, HeLa cells transfected with mouse EGFP-Myo18Aβ2, 1.5 × 105 purified peritoneal F4/80+ cells (macrophages) from WT mice, 1.5 × 105 purified peritoneal EYFP+ (Cre recombinase–positive) cells from Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mice, and (mouse) NIH-3T3 cells. A separate blot shows the relative intensities of Myo18A alternative splicing isoforms. D, relative protein levels of Myo18Aα (α) and Myo18Aβ (β) in purified mouse resident macrophages. Error bars, S.E. E, migration plots of WT and Myo18a-deficient macrophages in a complement C5a gradient. F, summary plots of chemotactic efficiency (chemotaxis index) and mean velocity (n = 100 cells (4 mice) in each group); n.s. = not significant at the 0.05 level of significance (Mann–Whitney U test). Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blotting analyses.

    Techniques Used: Knock-Out, Expressing, Mutagenesis, Activity Assay, Fluorescence, Confocal Microscopy, Isolation, Western Blot, Purification, Transfection, Alternative Splicing, Migration, Chemotaxis Assay, MANN-WHITNEY

    Myo18A does not localize to Golgi and Myo18a-deficient macrophages have normal Golgi morphology. A, resolution of the Zeiss Elyra superresolution structured illumination microscope (SR-SIM) determined by point spread function measurements. Subdiffraction-sized fluorescent beads (100 nm diameter) were imaged via a ×63 oil immersion objective lens (numerical aperture 1.46), and stacks were obtained at 110-nm steps. B, SR-SIM images of fixed WT and Myo18a-deficient (Myo18atm1c/tm1c/LysM-Cre; cKO) macrophages labeled with anti-GM130 (cis-Golgi) or anti-Giantin (Golgi membrane) antibodies (green) and the nucleic acid stain 4′,6-diamidino-2-phenylindole (blue). C, summary plots of Golgi area and Golgi extent (the fraction of the nuclear perimeter overlapped by Golgi cisternae) in WT versus Myo18a-deficient macrophages; n.s., not significant at the 0.05 level of significance (Mann–Whitney U test). D, SR-SIM images of living HeLa cells stably transfected with Myo18A-EGFP (green) and transiently transfected with the Golgi probe RFP-Golgi (red). Right, box plot of Pearson's correlation coefficient. Scale bar, 10 μm.
    Figure Legend Snippet: Myo18A does not localize to Golgi and Myo18a-deficient macrophages have normal Golgi morphology. A, resolution of the Zeiss Elyra superresolution structured illumination microscope (SR-SIM) determined by point spread function measurements. Subdiffraction-sized fluorescent beads (100 nm diameter) were imaged via a ×63 oil immersion objective lens (numerical aperture 1.46), and stacks were obtained at 110-nm steps. B, SR-SIM images of fixed WT and Myo18a-deficient (Myo18atm1c/tm1c/LysM-Cre; cKO) macrophages labeled with anti-GM130 (cis-Golgi) or anti-Giantin (Golgi membrane) antibodies (green) and the nucleic acid stain 4′,6-diamidino-2-phenylindole (blue). C, summary plots of Golgi area and Golgi extent (the fraction of the nuclear perimeter overlapped by Golgi cisternae) in WT versus Myo18a-deficient macrophages; n.s., not significant at the 0.05 level of significance (Mann–Whitney U test). D, SR-SIM images of living HeLa cells stably transfected with Myo18A-EGFP (green) and transiently transfected with the Golgi probe RFP-Golgi (red). Right, box plot of Pearson's correlation coefficient. Scale bar, 10 μm.

    Techniques Used: Microscopy, Labeling, Membrane, Staining, MANN-WHITNEY, Stable Transfection, Transfection

    Related Articles

    Western Blot:

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart
    Article Snippet: .. Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blotting analyses. ..

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart
    Article Snippet: .. Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, United Kingdom), diluted 1:1000, was used to detect mouse Myo18A protein by Western blot. ..

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart
    Article Snippet: .. Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting. ..

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart
    Article Snippet: .. Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blot analyses. ..



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    Homozygous <t>Myo18a</t> deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.
    Rabbit Polyclonal Anti Myo18a Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies rabbit polyclonal anti-myo18a  (Proteintech)
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    Proteintech antibodies rabbit polyclonal anti-myo18a
    Homozygous <t>Myo18a</t> deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.
    Antibodies Rabbit Polyclonal Anti Myo18a, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+polyclonal+anti+myo18a+antibody/rabbit+anti+myosin+18a+antibodies/pm27641956-332-2-33
    Average 90 stars, based on 1 article reviews
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    Image Search Results


    KEY RESOURCES TABLE

    Journal: Current biology : CB

    Article Title: Tyrosine phosphorylation of the myosin regulatory light chain controls non-muscle myosin II assembly and function in migrating cells

    doi: 10.1016/j.cub.2020.04.057

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-Myo18A , Novus Biologicals , Cat#NB100–79839.

    Techniques: Magnetic Beads, Virus, Recombinant, Membrane, Protease Inhibitor, Plasmid Preparation, Software, Mass Spectrometry, Chemotaxis Assay, Migration, Pore Size

    KEY RESOURCES TABLE

    Journal: Cell

    Article Title: The Golgi Outpost Protein TPPP Nucleates Microtubules and is Critical for Myelination

    doi: 10.1016/j.cell.2019.08.025

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Anti-MYO18A, rabbit polyclonal , Sigma , Cat#HPA021121.

    Techniques: Western Blot, Staining, Recombinant, Affinity Chromatography, Fluorescence, Immunoprecipitation, Sequencing, Modification, Knock-Out, Plasmid Preparation, Software, Imaging, Microscopy, Electron Microscopy, Transferring

    Homozygous Myo18a deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Homozygous Myo18a deletion is lethal. A and B, schematic diagram of the Myo18a knockout first targeting strategy and conversion of the tm1a (knockout first) allele to tm1c (floxed) and tm1d (knockout) alleles, respectively. Germ line deletion of Myo18a was produced by interbreeding Myo18atm1c and PGK-Cre mice (to yield Myo18atm1d). Myo18a targeting was confirmed by Southern blot analysis. C, PCR genotyping of offspring derived from crossing HET knockout first (Myo18a+/tm1a × Myo18a+/tm1a) mice. Notably, no mice HOM for the knockout first allele were detected. D, PCR genotyping of offspring derived from crossing heterozygous floxed (Myo18a+/tm1c × Myo18a+/tm1c) or heterozygous Myo18a knockout (Myo18a+/tm1d × Myo18a+/tm1d) mice. E, no homozygous Myo18a knockout (Myo18atm1d/tm1d) mice were detected at E13.5, indicating that Myo18a deletion is embryonic lethal.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Knock-Out, Produced, Southern Blot, Derivative Assay

    X-gal staining and histology of Myo18atm1b embryos. A, conversion of the tm1a (knockout first) allele to the tm1b (reporter knockout) allele by crossing heterozygous knockout first (Myo18a+/tm1a) mice with ACTB-Cre mice. B, timing of embryonic lethality by timed intercrossings of heterozygous knockout reporter (Myo18a+/tm1b) mice. No viable homozygous embryos were detected after E12.5. *, data from crossing Myo18a+/tm1a × Myo18a+/tm1a. C, whole-mount X-gal staining of HET Myo18a reporter knockout embryos at E11.5 and E10.5. Scale bars, 1 mm. D, histological sections of the heart obtained from X-gal–stained WT and HOM Myo18a reporter knockout E10.5 embryos. Arrows, X-gal staining of myocardial cells. Scale bars, 250 μm. AVC, atrioventricular cushions; V, ventricle; A, atrium. E, hematoxylin and eosin (HE) staining of sagittal sections of E10.5 HOM knockout (Myo18atm1b/tm1b) and WT embryos. OFT, outflow tract; AVC, atrioventricular cushions; V, ventricle; A, atrium.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: X-gal staining and histology of Myo18atm1b embryos. A, conversion of the tm1a (knockout first) allele to the tm1b (reporter knockout) allele by crossing heterozygous knockout first (Myo18a+/tm1a) mice with ACTB-Cre mice. B, timing of embryonic lethality by timed intercrossings of heterozygous knockout reporter (Myo18a+/tm1b) mice. No viable homozygous embryos were detected after E12.5. *, data from crossing Myo18a+/tm1a × Myo18a+/tm1a. C, whole-mount X-gal staining of HET Myo18a reporter knockout embryos at E11.5 and E10.5. Scale bars, 1 mm. D, histological sections of the heart obtained from X-gal–stained WT and HOM Myo18a reporter knockout E10.5 embryos. Arrows, X-gal staining of myocardial cells. Scale bars, 250 μm. AVC, atrioventricular cushions; V, ventricle; A, atrium. E, hematoxylin and eosin (HE) staining of sagittal sections of E10.5 HOM knockout (Myo18atm1b/tm1b) and WT embryos. OFT, outflow tract; AVC, atrioventricular cushions; V, ventricle; A, atrium.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Staining, Knock-Out

    Mouse ventricular myocytes express a novel isoform of Myo18A and cardiac-restricted Myo18a deletion is lethal. A, generation of cardiac-restricted Myo18a knockout mice. Mice harboring the floxed Myo18a allele (Myo18atm1c) were crossed with Tnnt2-Cre mice, in which Cre recombinase is expressed in cardiac myocytes. Conditional knockout mice were further crossed with Cre reporter (R26R-EYFP) mice to derive triple mutant (cardiac-restricted Myo18a knockout with Cre reporter) mice. B, bright field and fluorescence images of ventricular myocytes isolated from a Myo18a+/tm1c/Tnnt2-Cre/R26R-EYFP mouse. Green (EYFP) fluorescence is an indicator of Cre recombinase activity. Scale bar, 10 μm. C, no homozygous cardiac-restricted Myo18a knockout (Myo18atm1c/tm1c/Tnnt2-Cre) mice were obtained after crossing Myo18atm1c/tm1c and Myo18a+/tm1c/Tnnt2-Cre mice, indicating that conditional deletion of Myo18a in cardiac myocytes is lethal. No Myo18atm1c/tm1c/Tnnt2-Cre embryos were detected at E13.5, and three dead embryos were detected at E14.5. D, photographs of HET and homozygous cardiac-restricted Myo18a knockout embryos at day E14.5. Scale bar, 0.5 mm. E, Western blotting analyses. A custom antibody against the last 18 amino acids of mouse Myo18A (anti-Myo18A-C-term) recognizes the Myo18A isoforms Myo18Aα and Myo18Aβ. Lysates were prepared from HeLa cells transiently expressing EGFP-Myo18Aα (mouse) or EGFP-Myo18Aβ (mouse). The custom anti-Myo18A-C-term antibody did not detect Myo18A in mouse heart lysate, whereas an antibody against the coiled-coil (CC) domain of Myo18A detected a larger than expected band on the same membrane.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Mouse ventricular myocytes express a novel isoform of Myo18A and cardiac-restricted Myo18a deletion is lethal. A, generation of cardiac-restricted Myo18a knockout mice. Mice harboring the floxed Myo18a allele (Myo18atm1c) were crossed with Tnnt2-Cre mice, in which Cre recombinase is expressed in cardiac myocytes. Conditional knockout mice were further crossed with Cre reporter (R26R-EYFP) mice to derive triple mutant (cardiac-restricted Myo18a knockout with Cre reporter) mice. B, bright field and fluorescence images of ventricular myocytes isolated from a Myo18a+/tm1c/Tnnt2-Cre/R26R-EYFP mouse. Green (EYFP) fluorescence is an indicator of Cre recombinase activity. Scale bar, 10 μm. C, no homozygous cardiac-restricted Myo18a knockout (Myo18atm1c/tm1c/Tnnt2-Cre) mice were obtained after crossing Myo18atm1c/tm1c and Myo18a+/tm1c/Tnnt2-Cre mice, indicating that conditional deletion of Myo18a in cardiac myocytes is lethal. No Myo18atm1c/tm1c/Tnnt2-Cre embryos were detected at E13.5, and three dead embryos were detected at E14.5. D, photographs of HET and homozygous cardiac-restricted Myo18a knockout embryos at day E14.5. Scale bar, 0.5 mm. E, Western blotting analyses. A custom antibody against the last 18 amino acids of mouse Myo18A (anti-Myo18A-C-term) recognizes the Myo18A isoforms Myo18Aα and Myo18Aβ. Lysates were prepared from HeLa cells transiently expressing EGFP-Myo18Aα (mouse) or EGFP-Myo18Aβ (mouse). The custom anti-Myo18A-C-term antibody did not detect Myo18A in mouse heart lysate, whereas an antibody against the coiled-coil (CC) domain of Myo18A detected a larger than expected band on the same membrane.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Knock-Out, Mutagenesis, Fluorescence, Isolation, Activity Assay, Western Blot, Expressing, Membrane

    Cloning and sequencing of the novel isoform of Myo18A expressed in mouse ventricular myocytes. A, representative Sashimi plot of data obtained by mouse ventricular myocyte RNA-Seq (n = 6 mice). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles (with dashed lines) indicate exons that are abundant in the ventricular myocyte transcript but absent in the aligned annotated genomic references corresponding to transcripts for Myo18Aα and Myo18Aβ. B, domain structure of the novel cardiac isoform of Myo18A, Myo18Aγ, as well as, for comparison, the domain structures of Myo18Aα and Myo18Aβ. The recognition sites of various anti-Myo18A antibodies used in this study are indicated below. Notably, the custom (anti-Myo18A-C-term) antibody is unable to recognize the novel C terminus of Myo18Aγ. PPII, polyproline II helix; KE, KE-rich region; PDZ, PDZ domain; IQ, IQ motif. C, alignment of the N-terminal protein sequence of Myo18Aγ, derived from cloning and sequencing cDNA from mouse ventricular myocytes, with a mouse protein sequence of 219 residues from the Ensembl database (MGP_FVBNJ_P0030691).

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Cloning and sequencing of the novel isoform of Myo18A expressed in mouse ventricular myocytes. A, representative Sashimi plot of data obtained by mouse ventricular myocyte RNA-Seq (n = 6 mice). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles (with dashed lines) indicate exons that are abundant in the ventricular myocyte transcript but absent in the aligned annotated genomic references corresponding to transcripts for Myo18Aα and Myo18Aβ. B, domain structure of the novel cardiac isoform of Myo18A, Myo18Aγ, as well as, for comparison, the domain structures of Myo18Aα and Myo18Aβ. The recognition sites of various anti-Myo18A antibodies used in this study are indicated below. Notably, the custom (anti-Myo18A-C-term) antibody is unable to recognize the novel C terminus of Myo18Aγ. PPII, polyproline II helix; KE, KE-rich region; PDZ, PDZ domain; IQ, IQ motif. C, alignment of the N-terminal protein sequence of Myo18Aγ, derived from cloning and sequencing cDNA from mouse ventricular myocytes, with a mouse protein sequence of 219 residues from the Ensembl database (MGP_FVBNJ_P0030691).

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Cloning, Sequencing, RNA Sequencing, Comparison, Derivative Assay

    Human homolog of mouse Myo18Aγ and mouse–human sequence alignment. A, representative Sashimi plot of data obtained by RNA-Seq analysis of human atrial tissue (n = 10 subjects). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles indicate exons that are detected in the human atrial tissue transcript but absent in the aligned genomic references corresponding to transcripts (a–f) for human MYO18A. The accession numbers are as follows: MYO18Aa (NM_078471), MYO18Ab (NM_203318), MYO18Ac (NM_001346765), MYO18Ad (NM_001346766), MYO18Ae (NM_001346767), and MYO18Af (NM_001346768). B, sequence alignment of the N and C termini (N- and C-term) of mouse (Mus musculus (Mus-mus)) Myo18Aγ with human (Homo sapiens (Hom-sap)) atrial MYO18A. Identical residues are highlighted in gray and indicated by an asterisk below the alignment. Conserved mutations (residues replaced by another amino acid with similar biochemical properties) are indicated by a colon, and semiconserved mutations are denoted by a period. The red numbers below the sequences for Homo sapiens correspond to the (red) numbered exons depicted in the Sashimi plot above.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Human homolog of mouse Myo18Aγ and mouse–human sequence alignment. A, representative Sashimi plot of data obtained by RNA-Seq analysis of human atrial tissue (n = 10 subjects). Exons are colored blue, and splice junction reads are depicted as arcs. The red rectangles indicate exons that are detected in the human atrial tissue transcript but absent in the aligned genomic references corresponding to transcripts (a–f) for human MYO18A. The accession numbers are as follows: MYO18Aa (NM_078471), MYO18Ab (NM_203318), MYO18Ac (NM_001346765), MYO18Ad (NM_001346766), MYO18Ae (NM_001346767), and MYO18Af (NM_001346768). B, sequence alignment of the N and C termini (N- and C-term) of mouse (Mus musculus (Mus-mus)) Myo18Aγ with human (Homo sapiens (Hom-sap)) atrial MYO18A. Identical residues are highlighted in gray and indicated by an asterisk below the alignment. Conserved mutations (residues replaced by another amino acid with similar biochemical properties) are indicated by a colon, and semiconserved mutations are denoted by a period. The red numbers below the sequences for Homo sapiens correspond to the (red) numbered exons depicted in the Sashimi plot above.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Sequencing, RNA Sequencing

    Subcellular localization of Myo18Aγ in neonatal rat ventricular cardiomyocytes. A, NRVMs were lipofected with expression plasmids encoding EGFP-tagged mouse Myo18Aγ (Myo18Aγ-EGFP; middle row) and mScarlet-I-tagged mouse Cypher 1c (middle column) or mScarlet-I–tagged rat Myl2 (right column). Alternatively, cells fixed 4 days post-transfection were labeled with antibodies directed against sarcomeric α-actinin (left column). Images were obtained by laser-scanning confocal microscopy. Scale bars, 10 μm. B, enlarged views of the rectangular regions of interest shown in the top row of A. A horizontal line was drawn across these regions of interest to obtain the corresponding density plots, shown below. a.u., arbitrary units.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Subcellular localization of Myo18Aγ in neonatal rat ventricular cardiomyocytes. A, NRVMs were lipofected with expression plasmids encoding EGFP-tagged mouse Myo18Aγ (Myo18Aγ-EGFP; middle row) and mScarlet-I-tagged mouse Cypher 1c (middle column) or mScarlet-I–tagged rat Myl2 (right column). Alternatively, cells fixed 4 days post-transfection were labeled with antibodies directed against sarcomeric α-actinin (left column). Images were obtained by laser-scanning confocal microscopy. Scale bars, 10 μm. B, enlarged views of the rectangular regions of interest shown in the top row of A. A horizontal line was drawn across these regions of interest to obtain the corresponding density plots, shown below. a.u., arbitrary units.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Expressing, Transfection, Labeling, Confocal Microscopy

    Ultrastructural analysis of Myo18a-deficient E10.5 embryo hearts. A and B, high-magnification images, obtained by transmission EM, showing well-defined sarcomeres in WT and HET Myo18a knockout (Myo18a+/tm1b) E10.5 embryo hearts. The insets indicate parallel bundled filaments. C, highly disorganized sarcomeres in HOM Myo18a knockout (Myo18atm1b/tm1b) E10.5 embryo hearts. Z, Z-disc; I, I-band; A, A-band. Scale bars, 250 nm.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Ultrastructural analysis of Myo18a-deficient E10.5 embryo hearts. A and B, high-magnification images, obtained by transmission EM, showing well-defined sarcomeres in WT and HET Myo18a knockout (Myo18a+/tm1b) E10.5 embryo hearts. The insets indicate parallel bundled filaments. C, highly disorganized sarcomeres in HOM Myo18a knockout (Myo18atm1b/tm1b) E10.5 embryo hearts. Z, Z-disc; I, I-band; A, A-band. Scale bars, 250 nm.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Transmission Assay, Knock-Out

    Myo18a and Myo18b are the predominantly expressed unconventional myosins in mouse ventricular myocytes, and Myo18Aγ is detected in both mouse cardiac and skeletal muscle. A, expression levels of unconventional myosins in mouse ventricular myocytes. Data were obtained by RNA sequence analysis (n = 6 mice), and expression levels are indexed as FPKM (fragments per kilobase of transcript per million mapped reads). B, expression levels of conventional (class II) myosins in mouse ventricular myocytes. Note that Myh6, the dominant cardiac-specific myosin heavy chain isoform in adult mice, is highly expressed (2 of the 6 samples exceeded the detection range; not shown). C, Western blot analysis. Anti-Myo18A coiled-coil domain antibodies were used to detect Myo18A isoforms expressed in HeLa cells, mouse heart, and mouse skeletal (gastrocnemius) muscle. Error bars, S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Myo18a and Myo18b are the predominantly expressed unconventional myosins in mouse ventricular myocytes, and Myo18Aγ is detected in both mouse cardiac and skeletal muscle. A, expression levels of unconventional myosins in mouse ventricular myocytes. Data were obtained by RNA sequence analysis (n = 6 mice), and expression levels are indexed as FPKM (fragments per kilobase of transcript per million mapped reads). B, expression levels of conventional (class II) myosins in mouse ventricular myocytes. Note that Myh6, the dominant cardiac-specific myosin heavy chain isoform in adult mice, is highly expressed (2 of the 6 samples exceeded the detection range; not shown). C, Western blot analysis. Anti-Myo18A coiled-coil domain antibodies were used to detect Myo18A isoforms expressed in HeLa cells, mouse heart, and mouse skeletal (gastrocnemius) muscle. Error bars, S.E.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Expressing, Sequencing, Western Blot

    Generation of myeloid-restricted Myo18a knockout mice and expression of the splice variants Myo18Aα and Myo18Aβ in macrophages. A, triple mutant Myo18a cKO mice. Shown is a schematic diagram of the floxed Myo18a allele (Myo18atm1c) and conversion to a myeloid-restricted Myo18a knockout allele with a fluorescent reporter (R26R-EYFP) of Cre recombinase activity. The bright field and fluorescence images on the right, obtained by spinning disk confocal microscopy, show resident peritoneal macrophages isolated from a Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mouse. Images are 120 × 120 μm. B, Western blot analysis of purified WT (F4/80+ cells) versus conditional Myo18a knockout macrophages (EYFP+ cells). In each lane, lysate from 2 × 105 cells was loaded. C, Western blot analysis of Myo18A protein detected in the following cell lysates: untransfected (human) HeLa cells, HeLa cells transfected with mouse EGFP-Myo18Aβ1, HeLa cells transfected with mouse EGFP-Myo18Aβ2, 1.5 × 105 purified peritoneal F4/80+ cells (macrophages) from WT mice, 1.5 × 105 purified peritoneal EYFP+ (Cre recombinase–positive) cells from Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mice, and (mouse) NIH-3T3 cells. A separate blot shows the relative intensities of Myo18A alternative splicing isoforms. D, relative protein levels of Myo18Aα (α) and Myo18Aβ (β) in purified mouse resident macrophages. Error bars, S.E. E, migration plots of WT and Myo18a-deficient macrophages in a complement C5a gradient. F, summary plots of chemotactic efficiency (chemotaxis index) and mean velocity (n = 100 cells (4 mice) in each group); n.s. = not significant at the 0.05 level of significance (Mann–Whitney U test). Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blotting analyses.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Generation of myeloid-restricted Myo18a knockout mice and expression of the splice variants Myo18Aα and Myo18Aβ in macrophages. A, triple mutant Myo18a cKO mice. Shown is a schematic diagram of the floxed Myo18a allele (Myo18atm1c) and conversion to a myeloid-restricted Myo18a knockout allele with a fluorescent reporter (R26R-EYFP) of Cre recombinase activity. The bright field and fluorescence images on the right, obtained by spinning disk confocal microscopy, show resident peritoneal macrophages isolated from a Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mouse. Images are 120 × 120 μm. B, Western blot analysis of purified WT (F4/80+ cells) versus conditional Myo18a knockout macrophages (EYFP+ cells). In each lane, lysate from 2 × 105 cells was loaded. C, Western blot analysis of Myo18A protein detected in the following cell lysates: untransfected (human) HeLa cells, HeLa cells transfected with mouse EGFP-Myo18Aβ1, HeLa cells transfected with mouse EGFP-Myo18Aβ2, 1.5 × 105 purified peritoneal F4/80+ cells (macrophages) from WT mice, 1.5 × 105 purified peritoneal EYFP+ (Cre recombinase–positive) cells from Myo18atm1c/tm1c/LysM-Cre/R26R-EYFP mice, and (mouse) NIH-3T3 cells. A separate blot shows the relative intensities of Myo18A alternative splicing isoforms. D, relative protein levels of Myo18Aα (α) and Myo18Aβ (β) in purified mouse resident macrophages. Error bars, S.E. E, migration plots of WT and Myo18a-deficient macrophages in a complement C5a gradient. F, summary plots of chemotactic efficiency (chemotaxis index) and mean velocity (n = 100 cells (4 mice) in each group); n.s. = not significant at the 0.05 level of significance (Mann–Whitney U test). Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe) was used for the above Western blotting analyses.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Knock-Out, Expressing, Mutagenesis, Activity Assay, Fluorescence, Confocal Microscopy, Isolation, Western Blot, Purification, Transfection, Alternative Splicing, Migration, Chemotaxis Assay, MANN-WHITNEY

    Myo18A does not localize to Golgi and Myo18a-deficient macrophages have normal Golgi morphology. A, resolution of the Zeiss Elyra superresolution structured illumination microscope (SR-SIM) determined by point spread function measurements. Subdiffraction-sized fluorescent beads (100 nm diameter) were imaged via a ×63 oil immersion objective lens (numerical aperture 1.46), and stacks were obtained at 110-nm steps. B, SR-SIM images of fixed WT and Myo18a-deficient (Myo18atm1c/tm1c/LysM-Cre; cKO) macrophages labeled with anti-GM130 (cis-Golgi) or anti-Giantin (Golgi membrane) antibodies (green) and the nucleic acid stain 4′,6-diamidino-2-phenylindole (blue). C, summary plots of Golgi area and Golgi extent (the fraction of the nuclear perimeter overlapped by Golgi cisternae) in WT versus Myo18a-deficient macrophages; n.s., not significant at the 0.05 level of significance (Mann–Whitney U test). D, SR-SIM images of living HeLa cells stably transfected with Myo18A-EGFP (green) and transiently transfected with the Golgi probe RFP-Golgi (red). Right, box plot of Pearson's correlation coefficient. Scale bar, 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

    doi: 10.1074/jbc.RA118.004560

    Figure Lengend Snippet: Myo18A does not localize to Golgi and Myo18a-deficient macrophages have normal Golgi morphology. A, resolution of the Zeiss Elyra superresolution structured illumination microscope (SR-SIM) determined by point spread function measurements. Subdiffraction-sized fluorescent beads (100 nm diameter) were imaged via a ×63 oil immersion objective lens (numerical aperture 1.46), and stacks were obtained at 110-nm steps. B, SR-SIM images of fixed WT and Myo18a-deficient (Myo18atm1c/tm1c/LysM-Cre; cKO) macrophages labeled with anti-GM130 (cis-Golgi) or anti-Giantin (Golgi membrane) antibodies (green) and the nucleic acid stain 4′,6-diamidino-2-phenylindole (blue). C, summary plots of Golgi area and Golgi extent (the fraction of the nuclear perimeter overlapped by Golgi cisternae) in WT versus Myo18a-deficient macrophages; n.s., not significant at the 0.05 level of significance (Mann–Whitney U test). D, SR-SIM images of living HeLa cells stably transfected with Myo18A-EGFP (green) and transiently transfected with the Golgi probe RFP-Golgi (red). Right, box plot of Pearson's correlation coefficient. Scale bar, 10 μm.

    Article Snippet: Rabbit polyclonal anti-Myo18A antibody (14611-1-AP; Proteintech Europe, Manchester, UK), diluted 1:1000, was used to detect mouse Myo18A protein by Western blotting.

    Techniques: Microscopy, Labeling, Membrane, Staining, MANN-WHITNEY, Stable Transfection, Transfection