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  • 99
    Millipore na k atpase α1
    Multiple myosins co-immunoprecipitate recombinant Na + /K + <t>-ATPase</t> <t>α1</t> subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)
    Na K Atpase α1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore na k atpase β2 subunit
    <t>Na,K-ATPase</t> β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained
    Na K Atpase β2 Subunit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore na k atpase alpha 1
    Representative immunofluorescence images of <t>Na,K-ATPase</t> <t>alpha-1,</t> -2, and −3 expression at the rat retina. Na,K-ATPase isoforms labeled with fluorescein in the left hand column images, vWF visualized with Texas red in the second column, nuclei are DAPI-labeled, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls when no primary antibody was used. Unique Na,K-ATPase alpha-1 -3 specific expression in the retinal cell layers is seen, similar to previous reports [ 23 , 44 ]. The arrow indicates alpha-2 expression in the capillaries at the retinal ganglion cell layer. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer are labeled for orientation on the alpha-3 DAPI image. Scale bar = 50 μm.
    Na K Atpase Alpha 1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Developmental Studies Hybridoma Bank mouse anti na k atpase α1 antibodies
    Multiple myosins co-immunoprecipitate recombinant Na + /K + <t>-ATPase</t> <t>α1</t> subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)
    Mouse Anti Na K Atpase α1 Antibodies, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Santa Cruz Biotechnology pan na k atpase α subunit antibody
    Multiple myosins co-immunoprecipitate recombinant Na + /K + <t>-ATPase</t> α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)
    Pan Na K Atpase α Subunit Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Developmental Studies Hybridoma Bank anti na k atpase α1 subunit antibodies
    Multiple myosins co-immunoprecipitate recombinant Na + /K + <t>-ATPase</t> <t>α1</t> subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)
    Anti Na K Atpase α1 Subunit Antibodies, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti na k atpase α1 subunit antibodies/product/Developmental Studies Hybridoma Bank
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    78
    Santa Cruz Biotechnology na k atpase β3 subunit
    <t>Na,K-ATPase</t> β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained
    Na K Atpase β3 Subunit, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher na k atpase β1 subunit
    <t>Na,K-ATPase</t> β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained
    Na K Atpase β1 Subunit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore sheep kidney na k atpase
    Stable expression of wild-type and mutant forms of rat α -1 subunit of Na+ /K+ <t>-ATPase</t> in OK cells
    Sheep Kidney Na K Atpase, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Becton Dickinson na k atpase β2 subunit
    <t>Na,K-ATPase</t> β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained
    Na K Atpase β2 Subunit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 76/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Western Blot

    Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Recombinant, Construct, Atomic Absorption Spectroscopy, Transfection, Immunoprecipitation, Western Blot

    Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot

    Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Binding Assay, Atomic Absorption Spectroscopy, Construct, Transfection, Immunoprecipitation, Western Blot

    Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Immunoprecipitation, Positive Control

    Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Immunoprecipitation

    Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Article Snippet: Immunoblots were incubated with monoclonal antibodies against myh9 (1:500; Abcam), myh10 (1:1000; Abcam), myosin regulatory light chain (MRLC) (1:200; Santa Cruz Biotechnology), Na+ /K+ -ATPase α1 (0.5 μg/mL; DSHB), Na+ /K+ -ATPase α1 (1:1000; EMD Millipore), Na+ /K+ -ATPase α (1:200; Santa Cruz Biotechnology), GFP (1:1000, NeuroMab), myosin VI (1:500; Sigma), voltage gated sodium channel α subunits (pan-Nav α; 1:1000; Sigma) and β-actin (1:10000; Sigma).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Staining

    Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Expressing, Purification, Staining, SDS Page

    Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Derivative Assay

    The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Western Blot, Immunoprecipitation

    The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques:

    Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Inhibition, Purification

    Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Incubation, Binding Assay

    Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Inhibition

    Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Staining

    Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Expressing, Purification, Staining, SDS Page

    Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Derivative Assay

    The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Western Blot, Immunoprecipitation

    The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques:

    Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Inhibition, Purification

    Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Incubation, Binding Assay

    Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Article Snippet: The polyclonal antibodies used were Na,K-ATPase α2 subunit (rabbit, 1:200; Millipore) and Na,K-ATPase β2 subunit (rabbit, 1:200; Millipore).

    Techniques: Inhibition

    Representative immunofluorescence images of Na,K-ATPase alpha-1, -2, and −3 expression at the rat retina. Na,K-ATPase isoforms labeled with fluorescein in the left hand column images, vWF visualized with Texas red in the second column, nuclei are DAPI-labeled, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls when no primary antibody was used. Unique Na,K-ATPase alpha-1 -3 specific expression in the retinal cell layers is seen, similar to previous reports [ 23 , 44 ]. The arrow indicates alpha-2 expression in the capillaries at the retinal ganglion cell layer. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer are labeled for orientation on the alpha-3 DAPI image. Scale bar = 50 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Representative immunofluorescence images of Na,K-ATPase alpha-1, -2, and −3 expression at the rat retina. Na,K-ATPase isoforms labeled with fluorescein in the left hand column images, vWF visualized with Texas red in the second column, nuclei are DAPI-labeled, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls when no primary antibody was used. Unique Na,K-ATPase alpha-1 -3 specific expression in the retinal cell layers is seen, similar to previous reports [ 23 , 44 ]. The arrow indicates alpha-2 expression in the capillaries at the retinal ganglion cell layer. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer are labeled for orientation on the alpha-3 DAPI image. Scale bar = 50 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Immunofluorescence, Expressing, Labeling, Gas Chromatography

    Representative images of meningeal areas showing DAB-stained Na,K-ATPase alpha-1, alpha-3, CGRP, and control, as indicated in the sections. M: branch of middle meningeal artery; N: trigeminal nerve fiber; c: capillary. Scale bar = 50 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Representative images of meningeal areas showing DAB-stained Na,K-ATPase alpha-1, alpha-3, CGRP, and control, as indicated in the sections. M: branch of middle meningeal artery; N: trigeminal nerve fiber; c: capillary. Scale bar = 50 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Staining

    Specificity of Na,K-ATPase alpha-2 immunoreactivity at the choroid plexus. The CP was labeled with anti-alpha-2 ( A ) or with peptide pre-absorbed anti-alpha-2 ( B ). The subtraction image is in C . D : no primary control. Scale bar = 20 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Specificity of Na,K-ATPase alpha-2 immunoreactivity at the choroid plexus. The CP was labeled with anti-alpha-2 ( A ) or with peptide pre-absorbed anti-alpha-2 ( B ). The subtraction image is in C . D : no primary control. Scale bar = 20 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Labeling

    Fluorescence images of Na,K-ATPase alpha-3 and CGRP in meninges. In the top row, Na,K-ATPase alpha-3 is labeled with fluorescein in the left, CGRP is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the arrowhead triplet indicate a trigeminal nerve. The lower horizontal images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-3 is expressed at the nerve fiber, not on the capillary. Scale bar = 10 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Fluorescence images of Na,K-ATPase alpha-3 and CGRP in meninges. In the top row, Na,K-ATPase alpha-3 is labeled with fluorescein in the left, CGRP is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the arrowhead triplet indicate a trigeminal nerve. The lower horizontal images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-3 is expressed at the nerve fiber, not on the capillary. Scale bar = 10 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Fluorescence, Labeling, Staining

    Representative immunofluorescence images of a choroid plexus from the 3rd ventricle, sequentially acquired at 1 μm steps in a z-stack to reveal the cellular location of Na,K-ATPase alpha-1 and −2 isoforms. The same Na,K-ATPase expression patterns were found in the choroid plexuses in the lateral ventricles (data not shown). Texas red is used to visualize alpha-1 and fluorescein for alpha-2, and the nuclei are DAPI stained. Negative controls (without primary antibody; data not shown) had no membrane or cytoplasmic staining. Scale bar = 10 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Representative immunofluorescence images of a choroid plexus from the 3rd ventricle, sequentially acquired at 1 μm steps in a z-stack to reveal the cellular location of Na,K-ATPase alpha-1 and −2 isoforms. The same Na,K-ATPase expression patterns were found in the choroid plexuses in the lateral ventricles (data not shown). Texas red is used to visualize alpha-1 and fluorescein for alpha-2, and the nuclei are DAPI stained. Negative controls (without primary antibody; data not shown) had no membrane or cytoplasmic staining. Scale bar = 10 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Immunofluorescence, Expressing, Staining

    Representative immunofluorescence images of the blood-aqueous barrier at the rat ciliary body with Na,K-ATPase alpha-1, -2, and −3 isoform expression. Na,K-ATPase isoforms labeled with fluorescein in left hand column images, vWF visualized with Texas red in second column, nuclei are DAPI stained, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls with no primary antibody. Merged images show alpha-1 expressed on the PE, alpha-2 on the NPE, and alpha-2 expressed on ciliary body capillaries. The arrowhead points to the PE on the alpha-1 merged image, and the arrow points to the NPE on the alpha-2 merged image. Scale bar = 20 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Representative immunofluorescence images of the blood-aqueous barrier at the rat ciliary body with Na,K-ATPase alpha-1, -2, and −3 isoform expression. Na,K-ATPase isoforms labeled with fluorescein in left hand column images, vWF visualized with Texas red in second column, nuclei are DAPI stained, and the merged image for each horizontal panel is in the right hand column. Lower horizontal images are negative controls with no primary antibody. Merged images show alpha-1 expressed on the PE, alpha-2 on the NPE, and alpha-2 expressed on ciliary body capillaries. The arrowhead points to the PE on the alpha-1 merged image, and the arrow points to the NPE on the alpha-2 merged image. Scale bar = 20 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Immunofluorescence, Expressing, Labeling, Staining

    A is a tiled DAB image of Na,K-ATPase alpha-2 immunostaining at the rat meninges. The field of view is half of the meninges, extending from the transverse sinus (TS) to the superior sagittal sinus (SSS) centrally and the middle meningeal artery (MMA) laterally, with the trigeminal nerve fibers (TN) crossing the dura. Scale bar 500 μm. B : Enlarged region of meninges, magnified from the rectangle area in Figure 9 A, illustrating branches of the MMA, a TN, and a capillary (DCap). Scale bar = 50 μm. For control, see Figure 10 .

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: A is a tiled DAB image of Na,K-ATPase alpha-2 immunostaining at the rat meninges. The field of view is half of the meninges, extending from the transverse sinus (TS) to the superior sagittal sinus (SSS) centrally and the middle meningeal artery (MMA) laterally, with the trigeminal nerve fibers (TN) crossing the dura. Scale bar 500 μm. B : Enlarged region of meninges, magnified from the rectangle area in Figure 9 A, illustrating branches of the MMA, a TN, and a capillary (DCap). Scale bar = 50 μm. For control, see Figure 10 .

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Immunostaining

    Fluorescence images of CGRP and Na,K-ATPase alpha-2 in meninges. In the top row, CGRP is labeled with fluorescein on the left, Na,K-ATPase alpha-2 is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the smaller arrowhead indicates a trigeminal nerve. The lower row images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-2 is expressed at the capillary and nerve fiber. Scale bar = 10 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Fluorescence images of CGRP and Na,K-ATPase alpha-2 in meninges. In the top row, CGRP is labeled with fluorescein on the left, Na,K-ATPase alpha-2 is visualized with Texas red, nuclei are DAPI-stained, and the merged image is on the right. The large arrow indicates a capillary and the smaller arrowhead indicates a trigeminal nerve. The lower row images are negative controls when no primary antibody was used. The notable finding is that Na,K-ATPase alpha-2 is expressed at the capillary and nerve fiber. Scale bar = 10 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Fluorescence, Labeling, Staining

    Specificity of Na,K-ATPase alpha-2 immunoreactivity at the retina. The retina was stained with anti-alpha-2 ( A ) or with pre-absorbed anti-alpha-2 ( B ). Subtraction image is shown in C . D : no primary negative control. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer; asterisk: ganglion cell layer; small arrow: RPE. Scale bar = 20 μm.

    Journal: Fluids and Barriers of the CNS

    Article Title: Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat

    doi: 10.1186/2045-8118-10-14

    Figure Lengend Snippet: Specificity of Na,K-ATPase alpha-2 immunoreactivity at the retina. The retina was stained with anti-alpha-2 ( A ) or with pre-absorbed anti-alpha-2 ( B ). Subtraction image is shown in C . D : no primary negative control. GC: ganglion cell layer; IN: inner nuclear layer; ON: outer nuclear layer; asterisk: ganglion cell layer; small arrow: RPE. Scale bar = 20 μm.

    Article Snippet: Meninges, CPs from the lateral and 3rd ventricles, and eyes were labeled with antibodies specific for the Na,K-ATPase alpha-1, alpha-2, and alpha-3 isoforms (1:1,000 unless otherwise stated, Millipore, Billerica, MA), von Willebrand factor (vWF) (1:500 dilution from Millipore or 1:100 dilution from Lifespan Biosciences, Seattle, WA), calcitonin gene-related peptide (CGRP) (1:5000 in Figure , 1:1,000 dilution in all other occasions, Peninsula Laboratories, LLC, San Carlos, CA), and occludin (1:200 dilution from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Staining, Negative Control, Gas Chromatography

    Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Recombinant, Transfection, Immunoprecipitation, Western Blot

    Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Recombinant, Construct, Atomic Absorption Spectroscopy, Transfection, Immunoprecipitation, Western Blot

    Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Recombinant, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot

    Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Binding Assay, Atomic Absorption Spectroscopy, Construct, Transfection, Immunoprecipitation, Western Blot

    Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Immunoprecipitation, Positive Control

    Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Immunoprecipitation

    Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Immunoprecipitation, Liquid Chromatography

    Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Article Snippet: HEK293 cell lysates (In, lane 1) were precleared (PC, lane 2) with mouse IgG2a isotypes prior to immunoprecipitation (IP, lane 3) using mouse anti-Na+ /K+ -ATPase α1 antibodies (DSHB: a6F) of the IgG2a isotypes.

    Techniques: Immunoprecipitation, Liquid Chromatography

    Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Western Blot

    Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Recombinant, Construct, Atomic Absorption Spectroscopy, Transfection, Immunoprecipitation, Western Blot

    Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot

    Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Binding Assay, Atomic Absorption Spectroscopy, Construct, Transfection, Immunoprecipitation, Western Blot

    Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation, Positive Control

    Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation

    Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Western Blot

    Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Recombinant, Construct, Atomic Absorption Spectroscopy, Transfection, Immunoprecipitation, Western Blot

    Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot

    Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Binding Assay, Atomic Absorption Spectroscopy, Construct, Transfection, Immunoprecipitation, Western Blot

    Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation, Positive Control

    Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation

    Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Article Snippet: Use of a pan-Na+ /K+ -ATPase α subunit antibody (Santa Cruz Biotechnology: sc-58,628/clone M7-PB-E9) for immunoblotting did not alter the outcome seen with the use of anti-Na+ /K+ -ATPase α1 subunit antibodies (DSHB) (Additional file : Figure S2B).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Staining

    Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Expressing, Purification, Staining, SDS Page

    Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Derivative Assay

    The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Western Blot, Immunoprecipitation

    The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques:

    Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition, Purification

    Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Incubation, Binding Assay

    Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition

    Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Staining

    Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Expressing, Purification, Staining, SDS Page

    Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Derivative Assay

    The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Western Blot, Immunoprecipitation

    The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques:

    Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Inhibition, Purification

    Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Incubation, Binding Assay

    Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Article Snippet: For immunofluorescent staining, the following monoclonal antibodies were used: Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:20; Millipore) and Na,K-ATPase β1 subunit (mouse, clone M17 P5 F11, 1:100; Affinity Bioreagents).

    Techniques: Inhibition

    Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Multiple myosins co-immunoprecipitate recombinant Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with mCherry (In, lane 1) or Na + /K + -ATPase α1 tagged with mCherry in the C-terminus (In, lane 4; Na + /K + ATPase α1-mCherry) were precleared (PC) with mouse IgG2b antibodies ( a and b ; lanes 2 and 5) or mouse IgG1 antibodies ( c ; lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-myh9 antibodies of the IgG2b isotypes ( a ; lanes 3 and 6), mouse anti-myh10 antibodies of the IgG2b isotypes ( b ; lanes 3 and 6) or mouse anti-myoVI antibodies of the IgG1 isotypes ( c ; lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. mCherry immunoreactive bands in lanes 4 and 6 but not in any other lanes indicated co-immunoprecipitation of recombinant Na + /K + -ATPase α1 subunits by myh9 (lane 6, panel (i)), myh10 (lane 6, panel (iii)) and myoVI (lane 6, panel (v)) from HEK293 cells transfected with Na + /K + -ATPase α1-mCherry plasmids but not from those transfected with mCherry. As expected β-actin (lanes 3 and 6 in panels (ii)) was co-immunoprecipitated with myh9. Myh10 and myoVI noticeably co-immunoprecipitated β-actin (lane 6 in (iv) and (vi)) from HEK293 cells overexpressing Na + /K + -ATPase α1 subunits but not from those overexpressing mCherry. Full length images of western blots are presented in Additional file 12 : Figure S11, Additional file 13 : Figure S12, Additional file 14 : Figure S13)

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Western Blot

    Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of full length or tail-less (ΔT) recombinant myo6 with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a and b Schematic representation of myo6 constructs. All the myo6 constructs possessed a short amino acid linker of 11 AAs (shown in blue colored letters in b ) between the mCherry tag (N-terminal) and coding sequences for myo6. mCherry tagged full length myo6 constructs (i.e., mCherry-Myo6) were truncated by 22 AAs (i.e., Δ1264–1285 = mCherry-myoVI-ΔT1), 60 AAs (i.e., Δ1226–1285 = mCherry-myoVI-ΔT2) and 120 AAs (i.e., Δ1166–1285 = mCherry-myoVI-ΔT3) from the C-terminal end where the numbers indicate the amino acid positions in the WT myo6. c Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with mCherry (In; 4), mCherry-myo6 (In; 7), mCherry-myo6-ΔT1 (In; 10), mCherry-myo6-ΔT2 (In; 13) or mCherry-myo6-ΔT3 (In; 16) plasmids (where mCherry tag is in the N-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11, 14 and 17) prior to immunoprecipitation using rabbit anti-mCherry antibodies (IP; lanes 3, 6, 9, 12, 15 and 18). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 12, 13, 15, 16 and 18 in (i) and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 14 and 17 in (i) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from the IP of recombinant mCherry-myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 but not from those of mCherry thus confirming interaction between myo6 and Na + /K + -ATPase α1 subunits which is not perturbed due to loss of tail regions in myo6. Recombinant mCherry-Myo6, mCherry-myo6-ΔT1, mCherry-myo6-ΔT2 or mCherry-myo6-ΔT3 could not co-immunoprecipitate β-actin (i.e., lanes 9, 12, 15 and 18 in (ii)) from HEK293 cells. Full length images of western blots are presented in Additional file 15 : Figure S14

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Recombinant, Construct, Atomic Absorption Spectroscopy, Transfection, Immunoprecipitation, Western Blot

    Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Recombinant myh9 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of HEK293 cells transiently transfected with GFP (In, lane 1) or myh9 tagged with GFP in the C-terminus (In, lane 4; myh9-GFP) were precleared (PC) with mouse IgG1 antibodies ( a , lanes 2 and 5) or goat immunoglobulins (gIgG) ( b , lanes 2 and 5) prior to immunoprecipitation (IP) using mouse anti-GFP antibodies of the IgG1 isotypes ( a , lanes 3 and 6) or goat anti-GFP antibodies ( b , lanes 3 and 6). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4 and 6 but not in any other lanes ((i) and (iii)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. As expected immunoprecipitation using anti-GFP antibodies led to the co-immunoprecipitation of β-actin (lane 6 in (ii) and (iv)) from HEK293 cells transfected with myh9-GFP plasmids but not from those transfected with GFP plasmids. Immunoprecipitation using goat anti-GFP antibodies ( b ) led to a cleaner co-IP of Na + /K + -ATPase α1 subunits from HEK293 cells expressing myh9-GFP. Full length images of western blots are presented in Figure Additional file 4 : Figure S3B (for part a ) and Additional file 5 : Figure S4B (for part b )

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Recombinant, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot

    Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of actin-binding-site-less (ΔABS) and tail-less (Δtail) NMHC-IIs with Na + /K + -ATPase α1 subunits expressed in HEK293 cells. a Tail-less NMHC-IIs were made by deleting the tail regions, yellow shaded, of human (h) myh9 (i.e., hMyh9 AAs: 1928–1960), human (h) myh10 isoform 2 (i.e., hMyh10.2 AAs: 1934–1976) and mouse (m) myh14 isoform 3 (i.e., mMyh14.3 AAs:1946–1992) following a conserved proline residue (bold and underlined) where the numbers indicate the position of the amino acids in the WT constructs. b Actin-binding-site-less (ΔABS) NMHC-IIs were made by deleting a 23 amino acids (AAs) segment, yellow shaded, of hMyh9 (i.e., AAs: 654–676), hMyh10.2 (i.e., AAs: 661–683) and mMyh14.3 (i.e., AAs: 674–696). For both ( a ) and ( b ) symbols below sequences indicate fully (*), strongly (:) or weakly (.) conserved residues, and the lack of a symbol indicates amino acid divergence. c The actin binding site (ABS) of human myh14. The 3-D coordinates of human myh14 was retrieved from protein data bank (PDB: 5I4E). Structural features of myh14 was visualized and analyzed using UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/ ). Left panel shows the relative position of the ABS (red ribbon), nucleotide analog (adenosine diphosphate vanadate (ADP.VO4): ball and stick model) binding site and N-terminal SH3 domain (deep blue) in the motor domain of human myh14 (5I4E.pdb). The center and right panel show the relative position of the ABS (red ribbon; and ball and stick model) with respect to the position of the adenosine diphosphate vanadate (ADP.VO4) and magnesium co-factor (green ball) in the motor domain of myh14. d Tail-less (Δtail) but not actin-binding-site-less (ΔABS) myh14 co-immunoprecipitate Na + /K + -ATPase α1 subunits expressed in HEK293 cells. Lysates of non-transfected HEK293 cells (In; 1) or HEK293 cells transiently transfected with GFP (In; 4), myh14-GFP (In; 7), myh14-ΔABS-GFP (In; 10), or myh14-Δtail-GFP (In; 13) plasmids (where the GFP tag is in their C-terminus) were precleared with rabbit IgG (PC; lanes 2, 5, 8, 11 and 14) prior to immunoprecipitation using rabbit anti-GFP antibodies (IP; lanes 3, 6, 9, 12 and 15). Loading of PC complexes in the gel preceded those of the IP complexes. Presence of Na + /K + -ATPase α1 immunoreactive bands in lanes 1, 4, 7, 9, 10, 13 and 15 and absence of any Na + /K + -ATPase α1 immunoreactive bands in lanes 2, 3, 5, 6, 8, 11, 12 and 14 (in (i)) indicated co-immunoprecipitation of Na + /K + -ATPase α1 subunits from HEK293 cells transfected with myh14-GFP or myh14-Δtail-GFP plasmids but not from non-transfected HEK293 cells or those transfected with GFP or myh14-ΔABS-GFP plasmids. While myh14-ΔABS-GFP showed almost complete loss of actin binding (indicated with double asterisk ‘*’; lane 12, panel (ii)) both myh14-GFP and myh14-Δtail-GFP co-immunoprecipitated β-actin (lane 9 and lane 15 in panel (ii) respectively). Full length images of western blots are presented in Additional file 7 : Figure S6 B

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Binding Assay, Atomic Absorption Spectroscopy, Construct, Transfection, Immunoprecipitation, Western Blot

    Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of multiple myosins with Na + /K + -ATPase α1 subunits expressed in rat brain. WT adult rat brain lysates (In, lane 1 in a and b ) were precleared (PC) with indicated immunoglobulin isotypes (PC; lane2 = mIgG2b, lane 6 = rIgG and lane 9 = mIgG1) prior to immunoprecipitation (IP) using indicated antibodies (IP; lane 3 = myh9, lane 4 = myh10, lane 5 = KIF5B, lane 7 = Myh14, lane 8 = myoVa and lane 10 = myoVI). Loading of PC complexes in the gel preceded those of the IP complexes. Na + /K + -ATPase α1 subunits (i) were co-immunoprecipitated with myh9, myh10, KIF5B, myh14, myoVa and myoVI expressed in rat brain tissues. Co-immunoprecipitation of Na + /K + -ATPase α1 subunits by KIF5B served as a positive control. All the myosins assayed co-immunoprecipitated β-actin (ii). Denatured mouse IgG-HC (i.e., lanes 2–5, 9 and 10; panel (ii)), but not those of rabbit IgG (i.e., lanes 6–8) separated from their intact immunoglobulins (that is used for PC or IP) could be seen as this section was probed with mouse anti-β-actin antibodies

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Immunoprecipitation, Positive Control

    Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Na + /K + -ATPase α1 subunits co-immunoprecipitate multiple myosins expressed in adult rat brain. WT adult rat brain lysates (In, lane 2 in a , b and c ) were precleared (PC) with mouse IgG1 antibodies (PC, lane 1 in a , b and c ) prior to immunoprecipitation (IP) using mouse anti-Na + /K + -ATPase α1 antibodies of the IgG1 isotypes (IP, lane 3 in a , b and c ). Loading of PC complexes in the gel preceded those of the lysate inputs (In). Na + /K + -ATPase α1 subunits co-immunoprecipitated myh9 ( a (i)), myh10 ( b , (i)), myoVa ( c (i)), β-actin ((iii) in a , b and c ) and myosin regulatory light chain (MRLC) ((iv) in a , b and c ) from rat brain. An asterisk (‘*’; (iv) in a , b and c ) indicates lack of detection of the input signal for the MRLCs. As expected anti-Na + /K + -ATPase α1 antibodies immunoprecipitated Na + /K + -ATPase α1 subunits expressed in brain tissues ((ii) in a , b and c )

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Immunoprecipitation

    Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of myosin Va (myoVa) and myosin VI (myoVI) with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with rabbit IgG ( a ) or mouse IgG1 ( b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myoVa ( a ) or myoVI ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myoVI ( b (iii)), but not those of myoVa ( a (iii)), led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) expressed in HEK293 cells. Neither myoVa nor myoVI co-immunoprecipitated β-actin ((ii) in a and b ) expressed in HEK293 cells. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in B as the blot sections were probed with mouse anti- Na + /K + ATPase α1 and anti-β-actin antibodies

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Journal: Molecular Brain

    Article Title: Multiple myosin motors interact with sodium/potassium-ATPase alpha 1 subunits

    doi: 10.1186/s13041-018-0388-1

    Figure Lengend Snippet: Interaction of non-muscle myosin heavy chains with Na + /K + -ATPase α1 subunits endogenously expressed in HEK293 cells. HEK293 cell lysates (In, lane 1 in a and b ) were precleared (PC) with mouse IgG2b (PC, lane 2 in a and b ) prior to immunoprecipitation (IP, lane 3) using antibodies for myh9 ( a ) and myh10 ( b ). Loading of PC complexes in the gel preceded those of the IP complexes. Immunoprecipitation of myh9 ( a ) and myh10 ( b ) led to the co-immunoprecipitation of Na + /K + ATPase α1 subunits ((i) in a and b ) and β-actin ((ii) in a and b ) expressed in HEK293 cells. Na + /K + -ATPase α1 and β-actin immunoreactive signals in the depleted supernatant lane (DS, lane 4 in a ) indicates that the ATPase survives the IP procedure. Denatured mouse IgG-HC and IgG-LC separated from their intact immunoglobulins (that is used for PC or IP) are seen in ( a and b ) as those blot sections were probed with mouse anti-β-actin antibodies

    Article Snippet: A mouse Na+ /K+ -ATPase α1 cDNA clone was obtained from Dharmacon (Lafayette, CO, USA).

    Techniques: Immunoprecipitation, Liquid Chromatography

    Stable expression of wild-type and mutant forms of rat α -1 subunit of Na+ /K+ -ATPase in OK cells

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Stable expression of wild-type and mutant forms of rat α -1 subunit of Na+ /K+ -ATPase in OK cells

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques: Expressing, Mutagenesis

    Relative effect of Ang II on the elution of plasma membrane protein containing Populations #1, #2 and #3 of Na + /K + -ATPase from rat α -1.wild-type (A), α -1.S11A/S18A (B) and α -1.S938A (C) cells

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Relative effect of Ang II on the elution of plasma membrane protein containing Populations #1, #2 and #3 of Na + /K + -ATPase from rat α -1.wild-type (A), α -1.S11A/S18A (B) and α -1.S938A (C) cells

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques:

    Three populations of Na+ /K+ -ATPase in plasma membranes of α -1.S11A/S18A and α -1.S938A cells

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Three populations of Na+ /K+ -ATPase in plasma membranes of α -1.S11A/S18A and α -1.S938A cells

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques:

    Phosphorylation of rat kidney Na + /K + -ATPase at Ser 18

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Phosphorylation of rat kidney Na + /K + -ATPase at Ser 18

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques:

    Phosphorylation of rat kidney Na + /K + -ATPase at Ser 11

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Phosphorylation of rat kidney Na + /K + -ATPase at Ser 11

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques:

    Phosphorylation of rat kidney Na + /K + -ATPase at Ser 938

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Phosphorylation of rat kidney Na + /K + -ATPase at Ser 938

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques:

    Elution from digoxin-affinity columns of plasma membrane protein containing Na + /K + -ATPase Populations #1 (fractions 1–9), #2 (fractions 10–18) and #3 (fractions 19–22) from control and Ang II-treated α -1.S11A/S18A cells

    Journal:

    Article Title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

    doi: 10.1042/BJ20111398

    Figure Lengend Snippet: Elution from digoxin-affinity columns of plasma membrane protein containing Na + /K + -ATPase Populations #1 (fractions 1–9), #2 (fractions 10–18) and #3 (fractions 19–22) from control and Ang II-treated α -1.S11A/S18A cells

    Article Snippet: All other reagents, including the antibody against the α -subunit of sheep kidney Na+/ K+ -ATPase (M8-P1-A3) were purchased from Sigma–Aldrich.

    Techniques:

    Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Na,K-ATPase β 2 and α 2 subunits are localized almost exclusively in T-tubules in cardiomyocytes, whereas the α 1 and β 1 subunits are localized in both sarcolemma and T-tubules. A , frozen sections of rat heart were double-stained

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Staining

    Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Expression of purified Na,K-ATPase isoforms. Coomassie-stained SDS-PAGE of purified isoforms (5 μg/lane). For deglycosylation, samples were denatured and treated with PNGase F for 60 min at 37 °C.

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Expressing, Purification, Staining, SDS Page

    Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Models for docking of DcB to α 1 β 1 , α 2 β 2 , and α 2 β 3 . DcB was docked into homology models of human Na,K-ATPase isoforms derived from the porcine E2P·Mg·digoxin structure (Protein Data Bank code

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Derivative Assay

    The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit preferentially interact with each other in mouse heart. A , Western blotting analysis of proteins immunoprecipitated and co-immunoprecipitated from the detergent extracts of mouse heart microsome

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Western Blot, Immunoprecipitation

    The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: The Na,K-ATPase α 2 subunit and β 2 subunit are preferentially expressed in the mouse embryonic heart in contrast to the ubiquitously expressed α 1 subunit. A , paraffin-embedded sections of mouse embryos (embryonic day 12.5) were

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques:

    Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-digoxin antagonism depicted for α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 . Inhibition of purified Na,K-ATPase isoforms by digoxin was measured, and K i values were plotted against the potassium

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition, Purification

    Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Potassium-[ 3 H]digoxin antagonism. Yeast membranes harboring α 2 β 1 , α 2 β 2 , or α 2 β 3 Na,K-ATPase were incubated with varying concentrations of KCl, and residual [ 3 H]digoxin binding was measured. The lines represent

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Incubation, Binding Assay

    Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Journal:

    Article Title: Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

    doi: 10.1074/jbc.M116.751735

    Figure Lengend Snippet: Inhibition of Na,K-ATPase Isoforms by Perhydro-1,4-oxazepine Derivatives of Digoxin

    Article Snippet: For Western blotting analysis, the following monoclonal antibodies were used: against the Na,K-ATPase α1 subunit (mouse, clone C464.6, 1:1000; Millipore), against the Na,K-ATPase β2 subunit (mouse, clone 35; BD Transduction Laboratories), and Na,K-ATPase β3 subunit (goat, 1:500; Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition