Journal: Frontiers in Medicine

Article Title: Construction of tissue engineered cornea with skin-derived corneal endothelial-like cell and mechanism research for the cell differentiation

doi: 10.3389/fmed.2024.1448248

Figure Lengend Snippet: SKPs differentiated into CEC-like cells. (A) SKPs were cultured as floating spheres. During cell differentiation, the morphology of the cells changed gradually. On day 10, the cells became the most endothelial-like and formed a mosaic monolayer. (B) Immunofluorescence showed that the CEC-like cells expressed CEC markers Na + /K + ATPase, ZO-1, and Pitx2 after 10 days of differentiation. (C) RT-PCR showed that the CEC-like cells expressed CEC markers Pax6, Cdh2, Car2, Slc4a4, Col4a2 and Col8a2. (Data are mean ± SEM, * p < 0.05, n = 3, scale bar = 100 μm).

Article Snippet: The primary antibodies used included Na + /K + ATPase (1:100, Novus, NB300-146), ZO-1 (1:100, CST, 13663S), PITX2 (1:100, Abcam, ab98297), and human nuclei (1,100, Millipore, MAB1281).

Techniques: Cell Culture, Cell Differentiation, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 2 Immunohistochemistry of a guinea pig choroid plexus (a, b) and a human ES obtained at surgery (c). Na/K-ATPase α1 and β3 subunits are expressed in the apical cell membrane (a). Framed area is magnified in b. The apical cell membrane (small arrow) ex- presses the α1 subunit, while the β3 isoform is strongly expressed in the perinuclear zone (long ar- row). An ES was labeled with antibodies against both β1 and β3 isoforms from different species (c). In the proximal part of the sac (left), the β1 isoform was more expressed and in the basolateral cell membrane of the epithelium. In the distal part (right), the β3 subunit was more expressed and in the apical cell membranes in the epithelium. Here, only a few cells express the β1 isoform (in- set). Left frame is magnified in Fig. 3

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Immunohistochemistry, Membrane, Labeling

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 3 Expression of Na/K-ATPase β1 isoform (SIM, single optical sec- tion) in the epithelium of the intraosseous part of the human ES. The basolateral cell membranes strongly express the transporter protein. Arrows mark an epithelial protrusion into the sac lumen (Lu). A capillary (cap) is seen in the loose connective tissue. Inset shows a cell expressing Na/K-ATPase at higher magnification

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Expressing

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 4 Nanoscopic resolution of Na/K-ATPase β1 isoform and CA ex- pression (single optical section) in a cylindrical cell the intermediate por- tion of the human ES. Inset showing level of sectioning (*) of the ES. There is a strong expression of Na/K-ATPase in the lateral/basolateral cell membrane (a). A part of the apical membrane also seems to express the β isoform (arrow). Higher magnification of protein aggregates in the cyto- sol bridging the space between the cell nucleus (Nu) and the lateral plas- ma membrane (b, c)

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Expressing, Membrane

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 6 SR-SIM (single optical section) of human ES (intermediate portion). Inset showing level of sectioning (*) of the ES. Expression of Na/K-ATPase α1 and β3 isoforms (a). There is strong co-labeling at the apical cell membrane in some epithelial cells (arrow at inset). High mag- nification of the apical cell membrane in a cell displayed in the framed area in c (b). The β3 isoform is also expressed in the cell nucleus (arrows) and apical cytoplasm near the cell membrane. Inset in c shows nanoscopic resolution of ion transporter isoforms in the apical cell membrane

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Expressing, Labeling, Membrane

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 5 SR-SIM (single optical section) of an epithelial villus in the human ES (intermediate portion). Inset showing level of sectioning (*) of the ES. Expression of Na/K-ATPase α1 isoform and epithelial sodium channel (ENaC). b. Strong expression of Na/K-ATPase is localized only to the apical cell membrane. c. Nanoscopic resolution shows molecular relation- ship between Na/K-ATPase and ENAC in the apical cytoplasm. d. ENAC fluorescence at the juxta-nuclear region (arrow)

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Expressing, Membrane, Fluorescence

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 7 SR-SIM (single optical section) of an epithelial villus in the intermediate portion of the human ES. Inset showing level of sectioning (*) of the ES. Epithelial cells co-express Na/K-ATPase α1 and β3 iso- forms in the apical cell membrane (a). A neighboring cell expresses only the β3 isoform. Inset shows framed area b at higher magnification. The nucleus contains α1 complexes (arrow), while the cytoplasm harbors both isoforms. The apical cell membrane framed (c) in a (b). Both α1 and β3 isoforms are expressed in the apical cytoplasm and coalesce in the cell membrane. Inset shows framed area at higher magnification

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Membrane

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 8 Confocal microscopy of the intermediate human ES showing expression of Na/K- ATPase β1 and NKCC2 (a). Many NKCC2-positive cells lack ATPase expression. Mineral cor- ticoid receptor expression in the ES (b). Both Na/K-ATPase-posi- tive and negative cells are posi- tive. SR-SIM showing heavy ac- tivity of Na/K-ATPase in the basolateral plasma membrane (filled arrow) and NKCC2 posi- tivity (c). Confocal microscopy show expression of NCC and ENaC (d, e)

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Confocal Microscopy, Expressing, Clinical Proteomics, Membrane

Journal: Cell and tissue research

Article Title: "Reversed polarization" of Na/K-ATPase-a sign of inverted transport in the human endolymphatic sac: a super-resolution structured illumination microscopy (SR-SIM) study.

doi: 10.1007/s00441-019-03106-7

Figure Lengend Snippet: Fig. 9 Proposed implication of a “reversed polarization” of Na/K-ATPase isoform expression in the epithelium of the human ES. Cylindrical cells (left) express αβ1 in the basolateral membrane, while the right cell ex- presses α1β3 in the apical plasma membrane. A high Na+ conductance in the apical plasma membrane is due to the presence of amiloride-sensitive Na+ channels (Kim et al. 2009). Na+ channels and Na/K-ATPase may be controlled by the hormone aldosterone (Furuta et al. 1999; Akiyama et al. 2010). It may also activate apical and basolateral K+ channels (Mori and Wu 1996; Wu and Mori 1996; Kim et al. 2015). A thiazide-sensitive Na/ Cl cotransporter was also detected in the apical membrane by Akiyama et al. (Akiyama et al. 2008)

Article Snippet: At high magnification, Na/K-ATPase protein was seen to translocate from organelles near the cell nucleus to *α1 subunit mostly co-expressed with β3 subunit Table 1 Antibodies used in the study Antibody Type Dilution Host Catalog number Producer Na/K-ATPase β1 Monoclonal 1:100 Mouse MA3-930 Thermo Fisher, Waltham, USA Na/K-ATPase β2 Polyclonal 1:50 Rabbit ANP-012 Alomone Labs, Jerusalem, Israel Na/K-ATPase β3 Polyclonal 1:200 Rabbit Abx111158 Abbexa, Cambridge, UK Na/K-ATPase α1′ Monoclonal 1:50 Mouse SC 21712 Santa Cruz Biotechnology, Dallas, USA Na/K-ATPase α1′′ Monoclonal 1:50 Mouse NB300-146 Novus, Littleton, USA Na/K-ATPase α2 Polyclonal 1:100 Rabbit OAAB03232 Aviva Systems Biology, San Diego, USA Na/K-ATPase α3 Monoclonal 1:50 Mouse NB300-540 Novus, Littleton, USA Carbonic anhydrase II Polyclonal 1:50 Rabbit HPA001550 Atlas Antibodies, Stockholm, Sweden NKCC2 Polyclonal 1:200 Rabbit OABB01331 Aviva Systems Biology, San Diego, USA Mineralocorticoid receptor′ Monoclonal 1:50 Mouse MA1-620 Thermo Fisher, Waltham, USA Mineralocorticoid receptor′′ Polyclonal 1:200 Rabbit PA5-81527 Thermo Fisher, Waltham, USA Vasopressin receptor type 2 Polyclonal 1:50 Rabbit Abx133128 Abbexa, Cambridge, UK ENaC Polyclonal 1:100 Rabbit PA1-920A Thermo Fisher, Waltham, USA Aquaporin-2 Polyclonal 1:50 Rabbit NB110-74682 Novus, Littleton, USA Aquaporin-4 Polyclonal 1:50 Rabbit AQP-004 Alomone Labs, Jerusalem, Israel Pendrin Polyclonal 1:50 Rabbit NBP1-60106 Novus, Littleton, USA NKCC1 Polyclonal 1:100 Rabbit AB59791 Abcam, Cambridge, UK NCC Polyclonal 1:100 Rabbit PA5-77816 Thermo Fisher, Waltham, USA the lateral plasma membrane (Fig. 4, insets).

Techniques: Expressing, Membrane, Clinical Proteomics

Journal: The journal of headache and pain

Article Title: Functional correlation of ATP1A2 mutations with phenotypic spectrum: from pure hemiplegic migraine to its variant forms.

doi: 10.1186/s10194-021-01309-4

Figure Lengend Snippet: Fig. 5 Pump current recordings of Na+/K+-ATPase transfected in HEK293T cells. a and b Continuous recordings with wild-type pumps in HEK293T cells. 1 µM ouabain was added to block endogenous pumps. The pump current was activated by superfusion of 20 mM K+ and inhibited by 10 µM ouabain. c Representative raw currents of WT (blue), G762S (yellow), R938P (green) and G615R (red) in response to 200 ms steps with + 20 mV increments from −100 to + 100 mV. d Coexpression of ATP1A2 (green) and ATP1B1 (red) in transfected HEK293T cells. e Ouabain-sensitive pump currents measured in the last 50 ms of test pulses (two-way ANOVA: P = 0.040) (amplitude at + 40 mV, one-way ANOVA compared with WT, n = 9; G762S, n = 10, P = 0.018; R938P, n = 8, P = 0.142; G615R, n = 10, P = 0.008)

Article Snippet: cDNA constructs Full-length human ATP1A2 (NM_000702) and ATP1B1 (NM_001677) cDNAs were purchased from OriGene (pCMV6-AT1A2-Entry, Cat#RC208606; pCMV6AT1B1-Entry, Cat#RC200500, OriGene Technologies, Inc., Rockville, USA) and subcloned into the vectors pEF1α-IRES-AcGFP1 and pEF1α-IRES-DsRed-Express2, respectively.

Techniques: Transfection, Blocking Assay

Journal: PLoS ONE

Article Title: Na + /K + -ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

doi: 10.1371/journal.pone.0016547

Figure Lengend Snippet: A , Spectral count-weighted tabulation of GO annotation by biological process. Proteins involved in transport (42%) and metabolism (19%) are highly expressed in the blood-labyrinth barrier. B , The sequence of ATP1A1 covered by identified peptides is outlined in yellow (45% coverage). The alkylated residues (green) were identified by iodoacetamide treatment. C , A representative MS/MS spectrum for peptide 630–645 of ATP1A1. Graphically displayed are the fragment ions identified by SEQUEST from the tandem mass spectrometry data. Mass-to-charge (m/z) values are indicated next to each peak, along with positions of y-ions (blue) and b-ions (red). Unmatched ions are displayed as black. D, Localization of ATP1A1 (green) in stria vascularis capillaries (red, antibody for collagen type IV). A merged image (Right) shows the distribution of ATP1A1 along the capillary. Data in all panels are representative of at least 3 separate experiments. (Scale bars: D , 20 µm).

Article Snippet: The purified recombinant proteins used in this study were: ATP1A1 (Cat# TP301009) from Origene Technologies, Inc. and PKCη (Cat# 10-782-55103) from Geneway Biotech, Inc.

Techniques: Sequencing, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: PLoS ONE

Article Title: Na + /K + -ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

doi: 10.1371/journal.pone.0016547

Figure Lengend Snippet: A and B , Co-immunoprecipitation using isolated stria vascularis capillary lysates demonstrates that ATP1A1 is in a complex with occludin. Preimmune goat IgG served as a negative control. C , Immunolabeling of stria vascularis capillaries for occludin (Left, red) and ATP1A1 (Middle, green). The merged image (Right) shows that occludin and ATP1A1 co-localization. D , Confocal images show increased TJ permeability in ouabain-treated tissues (Lower) compared to control tissues (Upper). Serum protein IgG (green, antibody for IgG (H+L) is outside the lumen of the capillary (red, antibody for collagen type IV). E , REE analysis confirmed the significantly increased permeability of ouabain-treated tissues (**P = 0.005<0.01). F , Immunoblot analysis shows occludin migrates as two bands, hyperphosphorylated α (∼70 kD) and dephosphorylated β (∼65 kD), in both control and ouabain-treated tissues. G , The ratio of α band to β band in ouabain-treated tissues was significantly increased (**P = 0.007<0.01). Values in E and G are mean ± SEM (n = 10). (Scale bars: D , 20 µm). REE: relative extension of serum protein IgG extravasation; DV: diameter of the vessels; EE: extension of IgG extravasation.

Article Snippet: The purified recombinant proteins used in this study were: ATP1A1 (Cat# TP301009) from Origene Technologies, Inc. and PKCη (Cat# 10-782-55103) from Geneway Biotech, Inc.

Techniques: Immunoprecipitation, Isolation, Negative Control, Immunolabeling, Permeability, Control, Western Blot

Journal: PLoS ONE

Article Title: Na + /K + -ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

doi: 10.1371/journal.pone.0016547

Figure Lengend Snippet: A and B , Co-immunoprecipitation using isolated stria vascularis capillary lysates shows that PKCη is in a complex with ATP1A1. Goat IgG served as a negative control. C , Protein-protein interaction analysis with purified ATP1A1 (250 ng) and PKCη (250 ng). Ouabain (10 µM) was added where indicated. Control lanes consisted of either anti-ATP1A1 antibody and purified PKCη or anti-PKCη antibody and purified ATP1A1. D , Ouabain inhibition of Na + /K + -ATPase activity causes increased PKCη activity in isolated stria vascularis capillaries (*P = 0.013<0.05). E and F , PKCηPS pretreatment significantly attenuated ouabain-induced occludin hyperphosphorylation compared to pretreatment with PKCηNC (* P = 0.011<0.05). G , REE analysis shows a significant attenuation in ouabain-induced permeability in PKCηPS pre-treated tissues (**P = 0.005<0.01). REE, relative extension of serum protein IgG extravasation; DV, diameter of the vessels; EE, extension of IgG extravasation. Values in D , F and G are mean ± SEM (n = 10).

Article Snippet: The purified recombinant proteins used in this study were: ATP1A1 (Cat# TP301009) from Origene Technologies, Inc. and PKCη (Cat# 10-782-55103) from Geneway Biotech, Inc.

Techniques: Immunoprecipitation, Isolation, Negative Control, Purification, Control, Inhibition, Activity Assay, Permeability