Structured Review

Developmental Studies Hybridoma Bank anti na k atpase α1 subunit
A. Increased Na + /K + <t>-ATPase</t> pumping activity in cardiac fibroblasts from caveolin-1 knockout mice [Cav-1(−/−)]. Primary fibroblasts were assayed for ouabain-sensitive Rb + uptake for 10 min after treatment with 20 μM monensin. Data are means ± SE of 5 different preparations. * p
Anti Na K Atpase α1 Subunit, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti na k atpase α1 subunit/product/Developmental Studies Hybridoma Bank
Average 88 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti na k atpase α1 subunit - by Bioz Stars, 2020-09
88/100 stars

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1) Product Images from "Alterations of Na+/K+-ATPase Function in Caveolin-1 Knockout Cardiac Fibroblasts"

Article Title: Alterations of Na+/K+-ATPase Function in Caveolin-1 Knockout Cardiac Fibroblasts

Journal: Journal of molecular and cellular cardiology

doi: 10.1016/j.yjmcc.2010.04.015

A. Increased Na + /K + -ATPase pumping activity in cardiac fibroblasts from caveolin-1 knockout mice [Cav-1(−/−)]. Primary fibroblasts were assayed for ouabain-sensitive Rb + uptake for 10 min after treatment with 20 μM monensin. Data are means ± SE of 5 different preparations. * p
Figure Legend Snippet: A. Increased Na + /K + -ATPase pumping activity in cardiac fibroblasts from caveolin-1 knockout mice [Cav-1(−/−)]. Primary fibroblasts were assayed for ouabain-sensitive Rb + uptake for 10 min after treatment with 20 μM monensin. Data are means ± SE of 5 different preparations. * p

Techniques Used: Activity Assay, Knock-Out, Mouse Assay

Related Articles

Incubation:

Article Title: Alterations of Na+/K+-ATPase Function in Caveolin-1 Knockout Cardiac Fibroblasts
Article Snippet: .. Membranes were blocked with 4% nonfat dry milk in Tris-buffered saline solution plus 0.05% Tween 20 for 1 h followed by incubation overnight at 4°C with one of the following primary monoclonal or polyclonal antibodies in blocking solution: anti-Na+ /K+ -ATPase α1 subunit (α6F, Developmental Studies Hybridoma Bank, University of Iowa, USA), anti-Na+ /K+ -ATPase α2 subunit (Millipore Corp., Billerica, MA, USA), anti-Na+ /K+ -ATPase α3 subunit (Affinity Bioreagents, Golden, CO, USA), anti-phosphoERK1/2, anti-ERK1/2 (clone C-14), anti-actin (clone C-11) and anti-caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). .. After 1 h incubation with horseradish peroxidase-conjugated anti-mouse, anti-goat or anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Immunoreactivity was detected using chemiluminescence (Pierce, USA) and scanned images were analyzed by ImageJ software (NIH, USA).

Blocking Assay:

Article Title: Alterations of Na+/K+-ATPase Function in Caveolin-1 Knockout Cardiac Fibroblasts
Article Snippet: .. Membranes were blocked with 4% nonfat dry milk in Tris-buffered saline solution plus 0.05% Tween 20 for 1 h followed by incubation overnight at 4°C with one of the following primary monoclonal or polyclonal antibodies in blocking solution: anti-Na+ /K+ -ATPase α1 subunit (α6F, Developmental Studies Hybridoma Bank, University of Iowa, USA), anti-Na+ /K+ -ATPase α2 subunit (Millipore Corp., Billerica, MA, USA), anti-Na+ /K+ -ATPase α3 subunit (Affinity Bioreagents, Golden, CO, USA), anti-phosphoERK1/2, anti-ERK1/2 (clone C-14), anti-actin (clone C-11) and anti-caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). .. After 1 h incubation with horseradish peroxidase-conjugated anti-mouse, anti-goat or anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Immunoreactivity was detected using chemiluminescence (Pierce, USA) and scanned images were analyzed by ImageJ software (NIH, USA).

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  • 88
    Developmental Studies Hybridoma Bank monoclonal anti α1 na k atpase subunit antibody
    Generation and characterization of CD2 expressing cells (A) Stable cell lines expressing either YFP (YFP-1 and YFP-2) or YFP tagged CD2 (CD2-1, CD2-2, CD2-4 and CD2-5), were created from pig kidney epithelial cell line LLC-PK1. Equal amount of cell lysates from all the seven cell lines were analyzed by Western blot for YFP expression. (B) Fluorescent images of two representative cell lines are shown (images were taken at the same scale). (C) <t>Na/K-ATPase</t> <t>α1</t> and β1 subunit expression. Total cell lysates from LLC PK1, YFP-1, CD2-1 and CD2-2 cell lines were analyzed for expression of α1 and β1 subunit of Na/K-ATPase. Representative western blots are shown and quantitative data are from at least three independent experiments. As loading control, α-tubulin was probed on the same blot. (D) Cells were grown up to 100% confluence and ouabain binding was measured according to protocol described in “Materials and Methods” section. The values are mean± SEM from at least three independent experiments.* p
    Monoclonal Anti α1 Na K Atpase Subunit Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti α1 na k atpase subunit antibody/product/Developmental Studies Hybridoma Bank
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti α1 na k atpase subunit antibody - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    88
    Developmental Studies Hybridoma Bank anti na k atpase α1 subunit
    A. Increased Na + /K + <t>-ATPase</t> pumping activity in cardiac fibroblasts from caveolin-1 knockout mice [Cav-1(−/−)]. Primary fibroblasts were assayed for ouabain-sensitive Rb + uptake for 10 min after treatment with 20 μM monensin. Data are means ± SE of 5 different preparations. * p
    Anti Na K Atpase α1 Subunit, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti na k atpase α1 subunit/product/Developmental Studies Hybridoma Bank
    Average 88 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    anti na k atpase α1 subunit - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    Generation and characterization of CD2 expressing cells (A) Stable cell lines expressing either YFP (YFP-1 and YFP-2) or YFP tagged CD2 (CD2-1, CD2-2, CD2-4 and CD2-5), were created from pig kidney epithelial cell line LLC-PK1. Equal amount of cell lysates from all the seven cell lines were analyzed by Western blot for YFP expression. (B) Fluorescent images of two representative cell lines are shown (images were taken at the same scale). (C) Na/K-ATPase α1 and β1 subunit expression. Total cell lysates from LLC PK1, YFP-1, CD2-1 and CD2-2 cell lines were analyzed for expression of α1 and β1 subunit of Na/K-ATPase. Representative western blots are shown and quantitative data are from at least three independent experiments. As loading control, α-tubulin was probed on the same blot. (D) Cells were grown up to 100% confluence and ouabain binding was measured according to protocol described in “Materials and Methods” section. The values are mean± SEM from at least three independent experiments.* p

    Journal: PLoS ONE

    Article Title: SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase

    doi: 10.1371/journal.pone.0142119

    Figure Lengend Snippet: Generation and characterization of CD2 expressing cells (A) Stable cell lines expressing either YFP (YFP-1 and YFP-2) or YFP tagged CD2 (CD2-1, CD2-2, CD2-4 and CD2-5), were created from pig kidney epithelial cell line LLC-PK1. Equal amount of cell lysates from all the seven cell lines were analyzed by Western blot for YFP expression. (B) Fluorescent images of two representative cell lines are shown (images were taken at the same scale). (C) Na/K-ATPase α1 and β1 subunit expression. Total cell lysates from LLC PK1, YFP-1, CD2-1 and CD2-2 cell lines were analyzed for expression of α1 and β1 subunit of Na/K-ATPase. Representative western blots are shown and quantitative data are from at least three independent experiments. As loading control, α-tubulin was probed on the same blot. (D) Cells were grown up to 100% confluence and ouabain binding was measured according to protocol described in “Materials and Methods” section. The values are mean± SEM from at least three independent experiments.* p

    Article Snippet: The monoclonal anti-α1 Na/K-ATPase subunit antibody (α6f) was obtained from Developmental Studies Hybridoma Bank at The University of Iowa (Iowa City, IA).

    Techniques: Expressing, Stable Transfection, Western Blot, Binding Assay

    (A) A schematic diagram of Na/K-ATPase with all the important domains is shown. CD2-second cytosolic domain, P domain-phosphorylation domain and N domain- nucleotide binding domain. (B) GST pull down analysis showing a dose-dependent interaction between CD2 and His-SH2SH3. The upper panel shows the Western blot of anti-His, and the lower panel shows Ponceau staining of GST and GST-CD2. A representative Western blot from three independent experiments is shown.

    Journal: PLoS ONE

    Article Title: SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase

    doi: 10.1371/journal.pone.0142119

    Figure Lengend Snippet: (A) A schematic diagram of Na/K-ATPase with all the important domains is shown. CD2-second cytosolic domain, P domain-phosphorylation domain and N domain- nucleotide binding domain. (B) GST pull down analysis showing a dose-dependent interaction between CD2 and His-SH2SH3. The upper panel shows the Western blot of anti-His, and the lower panel shows Ponceau staining of GST and GST-CD2. A representative Western blot from three independent experiments is shown.

    Article Snippet: The monoclonal anti-α1 Na/K-ATPase subunit antibody (α6f) was obtained from Developmental Studies Hybridoma Bank at The University of Iowa (Iowa City, IA).

    Techniques: Binding Assay, Western Blot, Staining

    A. Increased Na + /K + -ATPase pumping activity in cardiac fibroblasts from caveolin-1 knockout mice [Cav-1(−/−)]. Primary fibroblasts were assayed for ouabain-sensitive Rb + uptake for 10 min after treatment with 20 μM monensin. Data are means ± SE of 5 different preparations. * p

    Journal: Journal of molecular and cellular cardiology

    Article Title: Alterations of Na+/K+-ATPase Function in Caveolin-1 Knockout Cardiac Fibroblasts

    doi: 10.1016/j.yjmcc.2010.04.015

    Figure Lengend Snippet: A. Increased Na + /K + -ATPase pumping activity in cardiac fibroblasts from caveolin-1 knockout mice [Cav-1(−/−)]. Primary fibroblasts were assayed for ouabain-sensitive Rb + uptake for 10 min after treatment with 20 μM monensin. Data are means ± SE of 5 different preparations. * p

    Article Snippet: Membranes were blocked with 4% nonfat dry milk in Tris-buffered saline solution plus 0.05% Tween 20 for 1 h followed by incubation overnight at 4°C with one of the following primary monoclonal or polyclonal antibodies in blocking solution: anti-Na+ /K+ -ATPase α1 subunit (α6F, Developmental Studies Hybridoma Bank, University of Iowa, USA), anti-Na+ /K+ -ATPase α2 subunit (Millipore Corp., Billerica, MA, USA), anti-Na+ /K+ -ATPase α3 subunit (Affinity Bioreagents, Golden, CO, USA), anti-phosphoERK1/2, anti-ERK1/2 (clone C-14), anti-actin (clone C-11) and anti-caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Activity Assay, Knock-Out, Mouse Assay

    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 + : 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 + : 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

    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 + : Figure S3B (for part a : 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 + : Figure S3B (for part a : 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

    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 ). 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 + : 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 ). 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 + : 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

    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

    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

    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

    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