Journal: Molecular Vision

Article Title: KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses

doi:

Figure Lengend Snippet: KCC2 and NKCC1 expression in a 30-day-old mouse lens. A : KCC2 mAb immunofluorescence detection (400X magnification) in mouse lens corresponds to the arrowhead in panel D . The lens perimeter is at the left. B : Anti-KCC2b (400X magnification; inset plus ~ 2X photomagnification). KCC2 is detected at the fiber cell borders in the interior lens fiber cells in the inset photo. Mouse lens fiber cells are about 7 μm in their longest dimension. C : Anti-KCC2b (600×, 3X photomagnification) identifies expression in cortical fiber cells as they terminally differentiate to mature fiber cells (arrowheads indicate the sites of the unstained cell nuclei). D : KCC2 is detected in interior fiber cells in the mouse lens (100X). The asterisk identifies the corresponding position in panel E and identifies KCC2 expression in the lens interior. The arrow at the lower left indicates the lens perimeter. E : NKCC1 mAb (monoclonal Ab) detection of cortical fiber cells with little detection in the interior lens nucleus. F : Lower exposure photo showing anti-NKCC1 polyclonal antibody detection restricted to lens cortical fibers in the mouse lens (200X, original magnification). G : KCC2b lower exposure detects protein in the cortical fiber cells. H : No primary antibody control. I : KCC2 detected at a site corresponding to the arrowhead in panel D . J : Actin detected in the same view as in panel H . K : I , J merged images. L : Immunoblot identifies KCC2b expression in the total mouse lens protein samples.

Article Snippet: Antibodies were obtained from the following sources: KCC2, NKCC1, and GABBR1 mAbs from Neuromab/Antibodies Inc. (Davis, CA), anti-NKCC1 from Alamone Labs (Jerusalem, Israel), and anti-KCC2b, anti-GABAAβ3, and STEP mAb from Santa Cruz Biotechnologies.

Techniques: Expressing, Immunofluorescence, Control, Western Blot

Journal: Molecular Vision

Article Title: KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses

doi:

Figure Lengend Snippet: NKCC1 and KCC2b in situ immunofluorescence detection in young adult rabbit lens. A , B : NKCC1 mAb and anti-KCC2 antibody detection in young adult rabbit lens (100X). C – E : NKCC1 expression in the cortical fiber cells is decreased as fiber cells mature in the lens interior. KCC2 is detected in fiber cells closer to the lens center. Merged images C , D are shown in panel E (200X). F : KCC2b is detected at the fiber cell membrane borders in mature interior fiber cells (200X). G : KCC2b viewed at 600X magnification. Rabbit fiber cells are about 10 μm across in their longest dimension. The asterisks identify comparable positions in the panels.

Article Snippet: Antibodies were obtained from the following sources: KCC2, NKCC1, and GABBR1 mAbs from Neuromab/Antibodies Inc. (Davis, CA), anti-NKCC1 from Alamone Labs (Jerusalem, Israel), and anti-KCC2b, anti-GABAAβ3, and STEP mAb from Santa Cruz Biotechnologies.

Techniques: In Situ, Immunofluorescence, Expressing, Membrane

Journal: Frontiers in Immunology

Article Title: Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways

doi: 10.3389/fimmu.2018.02049

Figure Lengend Snippet: Expressions of p-NKCC1 in rat lung tissues and MLE-12 cells. (A) Representative images of p-NKCC1 immunofluorescence staining (FITC-labeled green; original magnification ×400) of rat lung. Nuclei were counterstained with DAPI (blue). (B) T-NKCC1 and p-NKCC1 expressions in MLE-12 cells determined by western blot analysis ( n = 5 per group). (C) Histogram of p-NKCC1 determined by flow cytometry in MLE-12 cells ( n = 3 per group). BMT attenuated the activation of epithelial p-NKCC1 in IR-ALI. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD. * P < 0.05 compared with the control group; # P < 0.05 compared with the HR group.

Article Snippet: The anti-phosphorylated NKCC1 and anti-NKCC1 antibodies were custom made by GeneTex.

Techniques: Immunofluorescence, Staining, Labeling, Western Blot, Flow Cytometry, Activation Assay, Control

Journal: Frontiers in Immunology

Article Title: Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways

doi: 10.3389/fimmu.2018.02049

Figure Lengend Snippet: The mechanisms of NKCC1 modulating alveolar fluid clearance and inflammation in IR-ALI. IR stress causes phosphorylation of NKCC1 and activation of TRAF6, which result in cell swelling and inflammation of alveolar epithelium. Inhibition of NKCC1 by bumetanide reciprocally modulates epithelial TRAF6 expression. This interaction suppresses downstream p38 MAPK and NF-κB pathways, which attenuates the reduction of AFC via upregulating α-ENaC expression and reduces the alveolar inflammation.

Article Snippet: The anti-phosphorylated NKCC1 and anti-NKCC1 antibodies were custom made by GeneTex.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Expressing