Journal: American Journal of Physiology - Cell Physiology

Article Title: Slc26a6 is an apical membrane anion exchanger that drives HCO 3 − -dependent fluid secretion in murine pancreatic acinar cells

doi: 10.1152/ajpcell.00257.2019

Figure Lengend Snippet: Slc26a6 localizes to the apical membrane of pancreatic acinar cells. Immunofluorescent staining shows Slc26a6 (cyan stain, black arrows) and Na+-K+-Cl− cotransporter 1 (Nkcc1, red stain, red arrows) expression in the mouse pancreas. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). A and C: Slc26a6 immunolabeling primarily localized to the apical membrane of acinar cells in the Slc26a6+/+ mouse pancreas. B and D: Slc26a6-positive signal was not observed in the pancreas of Slc26a6−/− mice, confirming the specificity of the Slc26a6 antibody. Nkcc1 was expressed at the basolateral membrane of acinar cells in the pancreas of both Slc26a6+/+ and Slc26a6−/− mice. E and F: Slc26a6 immunolabeling (E) overlapped with differential interference contrast (DIC) image (F). White arrows indicate ducts. Scale bars = 30 µm.

Article Snippet: Sections were blocked in 20% donkey serum for 30 min at room temperature (RT) and then incubated overnight at 4°C with a rabbit anti-Slc26a6 antibody ( 32 ) diluted at 1:50 and a goat anti-Na + -K + -Cl − cotransporter 1 (NKCC1) antibody (catalog no. sc-21545, lot no. J0217, RRID:AB_2188633; Santa Cruz Biotechnology, Dallas, TX) diluted at 1:100, as previously described in mouse salivary glands ( 21 ).

Techniques: Membrane, Staining, Expressing, Immunolabeling

Journal: The Journal of Physiology

Article Title: Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3

doi: 10.1113/jphysiol.2012.236919

Figure Lengend Snippet: A–C, Western blots of parental and AE2-KD cell lysate proteins (50 μg total protein) probed with antibodies against: A, CFTR and α-actin; B, NKCC1 and α-actin; or C, NBCe1 and tubulin (Tub). D, quantification of transporters, normalized to α-actin or tubulin and plotted as a percentage of the level in parental cells. CFTR expression was increased by 72% in AE2-KD cells (n= 3, P < 0.05). E, comparison of CFTR levels in Scramble-KD and AE2-KD cells, confirming increased CFTR expression. F, intracellular cAMP levels (mean ± SEM, n= 3 each condition) were lower in AE2-KD cells compared with both parental (†P < 0.05) and Scramble-KD (*P < 0.05) cells according to cAMP chemiluminescent immunoassays.

Article Snippet: The following antibodies were used: AE2 (SA6 rabbit polyclonal antibody against the COOH-terminal amino acids 1224–1237 of mouse AE2 ( Stuart-Tilley et al. 1994 ) and rabbit polyclonal, Ab42687; 2 μg ml −1 ; Abcam), CFTR (mAb 23C5, 1:50), NBC (rabbit polyclonal, AB3208; 1:500, Millipore), NKCC1 (goat polyclonal, SC-21545, 1:200, Santa Cruz Biotechnology), tubulin (mouse monoclonal, T9822; 1:5000; Sigma) and β-actin (goat polyclonal, SC-1615, 1:200; Santa Cruz).

Techniques: Western Blot, Expressing, Comparison

Journal: The FASEB Journal

Article Title: Genetic dissection reveals unexpected influence of ? subunits on KCNQ1 K + channel polarized trafficking in vivo

doi: 10.1096/fj.10-173682

Figure Lengend Snippet: Reversed Kcnq1 trafficking in PCs of Kcne2−/− mice. A) Cartoon of a KCNQ1–KCNE2 complex. B) Cartoon of a PC showing location of HKA, NKCC1, and the KCNQ1–KCNE2 channel. C) KCNQ1 immunostaining (IS) in Kcne2−/− gastric mucosa. Scale bar = 100 μm. D) KCNQ1 IS in Kcne2−/− gastric mucosa (black box from panel C). Arrowhead indicates basolateral KCNQ1 staining. E) KCNQ1 IS in Kcne2+/+ gastric mucosa (same scale as panel D). Arrowhead indicates diffuse KCNQ1 staining due to localization at the invaginated apical membrane. F–H) Top: exemplar IF colabeling of Kcne2+/+ and Kcne2−/− gastric glands as indicated. “Merged” indicates merged view of the 2 panels above; bottom merged panel shows expanded view of the boxed region in the top merged panel. Yellow indicates colocalization. Blue arrowheads, PC basolateral side; white arrowheads, PC apical side. Representative of results from ≥2 mice, 3–5 sections/mouse/genotype. Bottom: cartoons summarizing IF data. F) Kcnq1 (red) and HKA β subunit (green). G) Kcnq1 (red) and NKCC (green). H) HKA β subunit (red) and NKCC (green). Width of view (except bottom merge): 100 μm (F); 75 μm (G, H).

Article Snippet: The primary antibody incubation (3 h) was followed by 32 min incubation with biotinylated anti-mouse IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) for HKA β, 60 min incubation with biotinylated anti-goat IgG (Vectastain ABC kit) for NKCC1 (and for KCNQ1 for KCNE3 colocalization analysis), and biotinylated anti-rabbit or antibody at 1:200 dilution (Vectastain ABC kit) for KCNQ1 (for HKA and NKCC1 colocalization analysis).

Techniques: Immunostaining, Staining

Journal: The FASEB Journal

Article Title: Genetic dissection reveals unexpected influence of ? subunits on KCNQ1 K + channel polarized trafficking in vivo

doi: 10.1096/fj.10-173682

Figure Lengend Snippet: Kcne3 deletion restores apical trafficking of Kcnq1 in Kcne2−/− mice. A) Targeting strategy for genomic deletion of Kcne3 from mice. B) Agarose gels showing PCR genotyping of wild-type, heterozygous, and homozygous single-knockout Kcne2−/− and Kcne3−/− mice, and double-knockout Kcne2−/−Kcne3−/− mice. C) Western blots of Kcne3 protein in membrane fractions from colonic crypts of Kcne3+/+ and Kcne3−/− mice as indicated, using an in-house antibody raised against a Kcne3 N-terminal epitope (left panel), and a commercial antibody (Alomone) raised against a Kcne3 C-terminal epitope (right panel). Migration distance of molecular mass markers is indicated at right. Arrows indicate band at 37 kDa unique to wild-type tissue. D) qRT-PCR analysis of remodeling of the fundic Kcne expression profile by targeted deletion of both Kcne2 and Kcne3; expression level expressed as a ratio to that of reference gene GAPDH amplified in parallel each time; n = 10 mice/single gene deletion genotype; n = 5 mice/double gene deletion genotype. ND, not determined (for Kcne3, not measured in Kcne2−/−Kcne3−/− mice; for Kcne5, unable to detect signal conforming to quality controls as described in Materials and Methods). Error bars = sem. E, F) Top: exemplar IF colabeling of Kcne2+/+ Kcne3−/− and Kcne2−/−Kcne3−/− gastric glands as indicated. Bottom two IF panels are merged views of the 3 panels above; bottom merged panel shows expanded view of the boxed region in the top merged panel. Blue arrowheads, PC basolateral side; white arrowheads, PC apical side. Counterstained with DAPI (blue). Representative results from ≥2 mice, 3–5 sections/mouse/genotype. Bottom: cartoons summarizing IF data. E) Kcnq1 (green) and HKA β subunit (red). F) Kcnq1 (red) and NKCC1 (green). Width of view (except bottom merge): 100 μm (E); 50 μm (F).

Article Snippet: The primary antibody incubation (3 h) was followed by 32 min incubation with biotinylated anti-mouse IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) for HKA β, 60 min incubation with biotinylated anti-goat IgG (Vectastain ABC kit) for NKCC1 (and for KCNQ1 for KCNE3 colocalization analysis), and biotinylated anti-rabbit or antibody at 1:200 dilution (Vectastain ABC kit) for KCNQ1 (for HKA and NKCC1 colocalization analysis).

Techniques: Knock-Out, Double Knockout, Western Blot, Migration, Quantitative RT-PCR, Expressing, Amplification

Journal: The FASEB Journal

Article Title: Genetic dissection reveals unexpected influence of ? subunits on KCNQ1 K + channel polarized trafficking in vivo

doi: 10.1096/fj.10-173682

Figure Lengend Snippet: Remodeled Kcne3 forms basolateral complexes with Kcnq1 in Kcne2−/− PCs. A) Co-IPs showing complex formation of Kcne3 with Kcnq1 (left panel) but not with NKCC1 (right panel) from mouse fundic membrane fractions. IPs of fractions from Kcne2+/+ and Kcne2−/− mice were prepared using antibodies raised against Kcnq1 or NKCC1, and Western blots were performed with anti-Kcne3 antibody. Numbers indicate migration of molecular mass markers (kDa). Arrow indicates expected mature Kcne3 migration distance. Top bands are precipitated antibodies. Representative of n = 2 experiments/antibody, with each prep pooled from 3–5 mouse stomachs/genotype. B) Top: exemplar IF labeling of Kcne3 (red) and Kcnq1 (green) in Kcne2+/+ or Kcne2−/− gastric glands, or Kcne2+/+ colonic crypts, as indicated. Merged panels show merged views of the 2 panels above; bottom merged panel shows expanded view of the boxed region in the top merged panel. Yellow indicates colocalization. Width of view (except bottom merge): 100 μm. Representative of results from at least two mice, 3–5 sections/mouse/genotype. Bottom: cartoons summarizing IF data.

Article Snippet: The primary antibody incubation (3 h) was followed by 32 min incubation with biotinylated anti-mouse IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) for HKA β, 60 min incubation with biotinylated anti-goat IgG (Vectastain ABC kit) for NKCC1 (and for KCNQ1 for KCNE3 colocalization analysis), and biotinylated anti-rabbit or antibody at 1:200 dilution (Vectastain ABC kit) for KCNQ1 (for HKA and NKCC1 colocalization analysis).

Techniques: Western Blot, Migration, Labeling

Journal: Stem Cell Research & Therapy

Article Title: Anti-inflammatory and vasculogenic conditioning of peripheral blood mononuclear cells reinforces their therapeutic potential for radiation-injured salivary glands

doi: 10.1186/s13287-019-1414-7

Figure Lengend Snippet: Histologic analysis of the submandibular glands at 12 weeks after IR. a Hematoxylin and eosin staining (scale bar, 50 μm) (× 200). Yellow arrow, fibrosis area; green arrow, vacuolar degeneration. b Masson’s trichrome staining (scale bar, 200 μm) (× 40). Fibrosis areas are stained blue. c PAS staining (scale bar, 100 μm) (× 100). Acinar cells were stained red. d Detection of the co-transporter for NKCC1 (a marker of acinar cells). Red, NKCC1; blue, DAPI (scale bar, 50 μm) (× 200). e The percentages of fibrosis area per field (%) (** p < 0.01, * p < 0.05). f The percentage area of acinar cells per field (%) (** p < 0.01). g The percentage area of ductal portions per field (%) (** p < 0.01, * p < 0.05)

Article Snippet: Immunohistological staining was performed with rat anti-mouse SCFR/c-Kit/CD117 antibody (1:50; R&D Systems, Minneapolis, MN, USA), rabbit anti-mouse Sca-1/Ly6 antibody (1:50; Abcam, Cambridge, UK), rabbit anti-mouse CD31 antibody (1:50; Abcam), mouse anti-mouse pan-cytokeratin antibody (1:100; Abcam), and goat anti-mouse NKCC1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Marker

Journal: Molecular Pain

Article Title: Inhibition of NKCC1 in spinal dorsal horn and dorsal root ganglion results in alleviation of neuropathic pain in rats with spinal cord contusion

doi: 10.1177/17448069231159855

Figure Lengend Snippet: Inhibition of NKCC1 in DRGs. (a): The mRNA expression level of NKCC1 gene was detected by qRT-PCR. (b): The protein level of NKCC1 was detected by WB analysis. (c): NKCC1 protein concentration in the three groups using the BCA assay. (d): DAPI staining of DRGs section with outlined spinal nerve dorsal root area and a squared inset of observed ganglion. (e-f): Quantification of NKCC1 immunoreactivity (green) with representative immunofluorescence images of DRGs sections. DAPI, blue; scale bar, 50 μm.

Article Snippet: Slides were blocked with normal goat serum for 30 min at 37°C, incubated with antibody against NKCC1 (#85403, CST, USA) overnight at 4°C, rinsed with 0.01 M phosphate buffered saline (PBS) 3 min for three times each, incubated with FITC-(for NKCC1) conjugated goat anti-rabbit IgG antibodies for 30 min at 37°C, and co-stained with DAPI.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Protein Concentration, BIA-KA, Staining, Immunofluorescence

Journal: Molecular Pain

Article Title: Inhibition of NKCC1 in spinal dorsal horn and dorsal root ganglion results in alleviation of neuropathic pain in rats with spinal cord contusion

doi: 10.1177/17448069231159855

Figure Lengend Snippet: Inhibition of NKCC1 in spinal cords. (a): The mRNA expression level of NKCC1 gene was detected by qRT-PCR. (b): The protein level of NKCC1 was detected by WB analysis. (c): NKCC1 protein concentration in the three groups using the BCA assay. (d): DAPI staining of spinal cord section with outlined gray matter area and a squared inset of observed ventral horn. (e): Immunostaining of NKCC1 (green) in the T10 region of spinal cords in different groups. DAPI, blue; sale bar, 50 μm. (f): Quantification of NKCC1 immunoreactivity. n.s.: Not significant between the three groups.

Article Snippet: Slides were blocked with normal goat serum for 30 min at 37°C, incubated with antibody against NKCC1 (#85403, CST, USA) overnight at 4°C, rinsed with 0.01 M phosphate buffered saline (PBS) 3 min for three times each, incubated with FITC-(for NKCC1) conjugated goat anti-rabbit IgG antibodies for 30 min at 37°C, and co-stained with DAPI.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Protein Concentration, BIA-KA, Staining, Immunostaining