Structured Review

Developmental Studies Hybridoma Bank mouse anti na k atpase α1
MPZL2 Displayed a Distinct Localization in the Cochlear Organ of Corti and Stria Vascularis at P4 in Wild-Type Mice (A) MPZL2 (red) localizes in the organ of Corti in the basal region of Deiters cells (DCs) present below the three rows of outer hair cells (OHCs) and diffusely in inner hair cells (IHCs), OHCs, and DCs. (B) In a serial section, collagen IV (green) immunostaining marked basement membranes, including the basilar membrane (BM), thereby indicating the localization of MPZL2 at the DC-BM contact region. (C) In the stria vascularis, MPZL2 (red) immunostaining was observed predominantly in the basal cell (BC) region. (D) Co-immunostaining of MPZL2 and Na + -K + <t>ATPase</t> (green), a marker for marginal cells (MCs), confirms immunostaining of MPZL2 (red) in the basal cells. Cell nuclei were stained with DAPI (blue). Arrowheads point to IHC, whitehead arrows to OHCs, and arrows to DCs. The scale bar (A–D) repsresents 20 μm.
Mouse Anti Na K Atpase α1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti na k atpase α1/product/Developmental Studies Hybridoma Bank
Average 75 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse anti na k atpase α1 - by Bioz Stars, 2020-02
75/100 stars

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1) Product Images from "MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse"

Article Title: MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse

Journal: American Journal of Human Genetics

doi: 10.1016/j.ajhg.2018.05.011

MPZL2 Displayed a Distinct Localization in the Cochlear Organ of Corti and Stria Vascularis at P4 in Wild-Type Mice (A) MPZL2 (red) localizes in the organ of Corti in the basal region of Deiters cells (DCs) present below the three rows of outer hair cells (OHCs) and diffusely in inner hair cells (IHCs), OHCs, and DCs. (B) In a serial section, collagen IV (green) immunostaining marked basement membranes, including the basilar membrane (BM), thereby indicating the localization of MPZL2 at the DC-BM contact region. (C) In the stria vascularis, MPZL2 (red) immunostaining was observed predominantly in the basal cell (BC) region. (D) Co-immunostaining of MPZL2 and Na + -K + ATPase (green), a marker for marginal cells (MCs), confirms immunostaining of MPZL2 (red) in the basal cells. Cell nuclei were stained with DAPI (blue). Arrowheads point to IHC, whitehead arrows to OHCs, and arrows to DCs. The scale bar (A–D) repsresents 20 μm.
Figure Legend Snippet: MPZL2 Displayed a Distinct Localization in the Cochlear Organ of Corti and Stria Vascularis at P4 in Wild-Type Mice (A) MPZL2 (red) localizes in the organ of Corti in the basal region of Deiters cells (DCs) present below the three rows of outer hair cells (OHCs) and diffusely in inner hair cells (IHCs), OHCs, and DCs. (B) In a serial section, collagen IV (green) immunostaining marked basement membranes, including the basilar membrane (BM), thereby indicating the localization of MPZL2 at the DC-BM contact region. (C) In the stria vascularis, MPZL2 (red) immunostaining was observed predominantly in the basal cell (BC) region. (D) Co-immunostaining of MPZL2 and Na + -K + ATPase (green), a marker for marginal cells (MCs), confirms immunostaining of MPZL2 (red) in the basal cells. Cell nuclei were stained with DAPI (blue). Arrowheads point to IHC, whitehead arrows to OHCs, and arrows to DCs. The scale bar (A–D) repsresents 20 μm.

Techniques Used: Mouse Assay, Immunostaining, Marker, Staining, Immunohistochemistry

Related Articles

Mutagenesis:

Article Title: MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse
Article Snippet: Paragraph title: Histology and Immunohistochemistry of Cochleae from Wild-Type and Mpzl2 -Mutant Mice ... Rabbit anti-MPZL2, raised against the complete protein (11787-1-AP, Proteintech), goat anti-Collagen IV (1340-01, SouthernBiotech), and mouse anti-Na+ -K+ ATPase α1 (α6F, Developmental Studies Hybridoma Bank).

Immunohistochemistry:

Article Title: MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse
Article Snippet: Paragraph title: Histology and Immunohistochemistry of Cochleae from Wild-Type and Mpzl2 -Mutant Mice ... Rabbit anti-MPZL2, raised against the complete protein (11787-1-AP, Proteintech), goat anti-Collagen IV (1340-01, SouthernBiotech), and mouse anti-Na+ -K+ ATPase α1 (α6F, Developmental Studies Hybridoma Bank).

Mouse Assay:

Article Title: MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse
Article Snippet: Paragraph title: Histology and Immunohistochemistry of Cochleae from Wild-Type and Mpzl2 -Mutant Mice ... Rabbit anti-MPZL2, raised against the complete protein (11787-1-AP, Proteintech), goat anti-Collagen IV (1340-01, SouthernBiotech), and mouse anti-Na+ -K+ ATPase α1 (α6F, Developmental Studies Hybridoma Bank).

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    Developmental Studies Hybridoma Bank mouse monoclonal anti na k atpase α1 antibody
    Reduction of membrane cholesterol by Mβ-CD down-regulates <t>α1</t> <t>Na/K-ATPase.</t> LLC-PK1 cells were treated with 10 m m Mβ-CD in the serum-free medium at 37 °C for 1 h, washed, and then collected after the washed cells were cultured
    Mouse Monoclonal Anti Na K Atpase α1 Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti na k atpase α1 antibody/product/Developmental Studies Hybridoma Bank
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti na k atpase α1 antibody - by Bioz Stars, 2020-02
    77/100 stars
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    Reduction of membrane cholesterol by Mβ-CD down-regulates α1 Na/K-ATPase. LLC-PK1 cells were treated with 10 m m Mβ-CD in the serum-free medium at 37 °C for 1 h, washed, and then collected after the washed cells were cultured

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?1 Na/K-ATPase Expression by Cholesterol *

    doi: 10.1074/jbc.M110.204396

    Figure Lengend Snippet: Reduction of membrane cholesterol by Mβ-CD down-regulates α1 Na/K-ATPase. LLC-PK1 cells were treated with 10 m m Mβ-CD in the serum-free medium at 37 °C for 1 h, washed, and then collected after the washed cells were cultured

    Article Snippet: The antibodies and their sources are as follows: The mouse monoclonal anti-Na/K-ATPase α1 antibody (α6F) for Western blot analysis was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

    Techniques: Cell Culture

    Membrane cholesterol-regulated trafficking of α1 Na/K-ATPase is dependent on Src kinase activity. A , LLC-PK1 cells were pretreated with 1 μ m PP2 for 1 h before addition of 10 m m Mβ-CD for 3 h. Cells were fixed and immunostained

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?1 Na/K-ATPase Expression by Cholesterol *

    doi: 10.1074/jbc.M110.204396

    Figure Lengend Snippet: Membrane cholesterol-regulated trafficking of α1 Na/K-ATPase is dependent on Src kinase activity. A , LLC-PK1 cells were pretreated with 1 μ m PP2 for 1 h before addition of 10 m m Mβ-CD for 3 h. Cells were fixed and immunostained

    Article Snippet: The antibodies and their sources are as follows: The mouse monoclonal anti-Na/K-ATPase α1 antibody (α6F) for Western blot analysis was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

    Techniques: Activity Assay

    Compound U18666A decreases plasma membrane cholesterol and specifically down-regulates α1 Na/K-ATPase. A , LLC-PK1 cells were treated with different doses of U18666A for 24 h and subjected to filipin ( top row of upper panel ) and α1 immunostaining

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?1 Na/K-ATPase Expression by Cholesterol *

    doi: 10.1074/jbc.M110.204396

    Figure Lengend Snippet: Compound U18666A decreases plasma membrane cholesterol and specifically down-regulates α1 Na/K-ATPase. A , LLC-PK1 cells were treated with different doses of U18666A for 24 h and subjected to filipin ( top row of upper panel ) and α1 immunostaining

    Article Snippet: The antibodies and their sources are as follows: The mouse monoclonal anti-Na/K-ATPase α1 antibody (α6F) for Western blot analysis was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

    Techniques: Immunostaining

    The proposed model of the Src-dependent interplay among the α1 Na/K-ATPase, caveolin-1, and cholesterol. This interplay establishes a feed-forward signaling mechanism that allows cells to regulate cholesterol distribution and Na/K-ATPase expression.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?1 Na/K-ATPase Expression by Cholesterol *

    doi: 10.1074/jbc.M110.204396

    Figure Lengend Snippet: The proposed model of the Src-dependent interplay among the α1 Na/K-ATPase, caveolin-1, and cholesterol. This interplay establishes a feed-forward signaling mechanism that allows cells to regulate cholesterol distribution and Na/K-ATPase expression.

    Article Snippet: The antibodies and their sources are as follows: The mouse monoclonal anti-Na/K-ATPase α1 antibody (α6F) for Western blot analysis was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

    Techniques: Expressing

    Down-regulation of the α1 Na/K-ATPase in target organs of BALB/c npc nih /npc nih mice. A , representative Western blots on the Na/K-ATPase α1 subunit, insulin receptor β, and α-tubulin of liver samples from NPC1 +/+ and NPC1

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?1 Na/K-ATPase Expression by Cholesterol *

    doi: 10.1074/jbc.M110.204396

    Figure Lengend Snippet: Down-regulation of the α1 Na/K-ATPase in target organs of BALB/c npc nih /npc nih mice. A , representative Western blots on the Na/K-ATPase α1 subunit, insulin receptor β, and α-tubulin of liver samples from NPC1 +/+ and NPC1

    Article Snippet: The antibodies and their sources are as follows: The mouse monoclonal anti-Na/K-ATPase α1 antibody (α6F) for Western blot analysis was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

    Techniques: Mouse Assay, Western Blot

    Membrane cholesterol reduction leads to the endocytosis and degradation of α1 Na/K-ATPase. A , LLC-PK1 cells were treated with 10 μg/ml U18666A for 24 h. Total RNA was extracted, and quantitative RT-PCR was performed to probe α1

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?1 Na/K-ATPase Expression by Cholesterol *

    doi: 10.1074/jbc.M110.204396

    Figure Lengend Snippet: Membrane cholesterol reduction leads to the endocytosis and degradation of α1 Na/K-ATPase. A , LLC-PK1 cells were treated with 10 μg/ml U18666A for 24 h. Total RNA was extracted, and quantitative RT-PCR was performed to probe α1

    Article Snippet: The antibodies and their sources are as follows: The mouse monoclonal anti-Na/K-ATPase α1 antibody (α6F) for Western blot analysis was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa.

    Techniques: Quantitative RT-PCR

    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

    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

    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: 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

    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: 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

    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: 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

    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

    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

    MPZL2 Displayed a Distinct Localization in the Cochlear Organ of Corti and Stria Vascularis at P4 in Wild-Type Mice (A) MPZL2 (red) localizes in the organ of Corti in the basal region of Deiters cells (DCs) present below the three rows of outer hair cells (OHCs) and diffusely in inner hair cells (IHCs), OHCs, and DCs. (B) In a serial section, collagen IV (green) immunostaining marked basement membranes, including the basilar membrane (BM), thereby indicating the localization of MPZL2 at the DC-BM contact region. (C) In the stria vascularis, MPZL2 (red) immunostaining was observed predominantly in the basal cell (BC) region. (D) Co-immunostaining of MPZL2 and Na + -K + ATPase (green), a marker for marginal cells (MCs), confirms immunostaining of MPZL2 (red) in the basal cells. Cell nuclei were stained with DAPI (blue). Arrowheads point to IHC, whitehead arrows to OHCs, and arrows to DCs. The scale bar (A–D) repsresents 20 μm.

    Journal: American Journal of Human Genetics

    Article Title: MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse

    doi: 10.1016/j.ajhg.2018.05.011

    Figure Lengend Snippet: MPZL2 Displayed a Distinct Localization in the Cochlear Organ of Corti and Stria Vascularis at P4 in Wild-Type Mice (A) MPZL2 (red) localizes in the organ of Corti in the basal region of Deiters cells (DCs) present below the three rows of outer hair cells (OHCs) and diffusely in inner hair cells (IHCs), OHCs, and DCs. (B) In a serial section, collagen IV (green) immunostaining marked basement membranes, including the basilar membrane (BM), thereby indicating the localization of MPZL2 at the DC-BM contact region. (C) In the stria vascularis, MPZL2 (red) immunostaining was observed predominantly in the basal cell (BC) region. (D) Co-immunostaining of MPZL2 and Na + -K + ATPase (green), a marker for marginal cells (MCs), confirms immunostaining of MPZL2 (red) in the basal cells. Cell nuclei were stained with DAPI (blue). Arrowheads point to IHC, whitehead arrows to OHCs, and arrows to DCs. The scale bar (A–D) repsresents 20 μm.

    Article Snippet: Rabbit anti-MPZL2, raised against the complete protein (11787-1-AP, Proteintech), goat anti-Collagen IV (1340-01, SouthernBiotech), and mouse anti-Na+ -K+ ATPase α1 (α6F, Developmental Studies Hybridoma Bank).

    Techniques: Mouse Assay, Immunostaining, Marker, Staining, Immunohistochemistry