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

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: Intracellular MICA/B and ULBP2 co-localize with Golgi apparatus and show different mechanism of intracellular trafficking. (A) Subcellular localization of MICA/B and ULBP2 (green) in BC cell lines, and co-localization with calnexin (endoplasmic reticulum, ER) and GM130 (Golgi) (red). Nuclei are counterstained with DAPI (blue). White arrows indicate co-localization (yellow/orange staining). (B) Calnexin or GM130 co-localization with MICA/B or ULBP2 was quantified by ImageJ plug-in JACOP. Experiment was performed twice and analyzed in at least 6 fields for each condition. Statistical analysis was performed using Kruskall-Wallis for non-parametric distribution with Dunn’s multiple comparisons within organelles: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C) Western blotting analysis of NKG2DLs and GM130 in subcellular fractions: cytosol (C), membrane (M), nucleus (N) and cytoskeleton (CS) extracts. TATA-binding protein (TBP), β-Actin and Na + /K + -ATPase were used as housekeeping for each fraction.

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Staining, Western Blot, Membrane, Binding Assay