Journal: Scientific Reports

Article Title: Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

doi: 10.1038/srep36016

Figure Lengend Snippet: Representative confocal microscope images show extent of expression and subcellular localization of TMEM16A, SLC26A4, carbonic anhydrase 2 (CA2), SLC12A2, and ATP12A (images taken from BE37 cells; similar results were obtained from BE63 cells). Whenever permitted by the combination of primary antibodies, acetylated tubulin and MUC5AC were also stained as markers of ciliated and goblet cells, respectively. Bronchial epithelia were kept under control conditions or treated with IL-4 for 72 hrs. Larger images: xy sections (scale bar: 20 μm). Inset: xz sections (scale bar: 10 μm). Images with a different scale of view are shown in .

Article Snippet: The following primary antibodies and dilutions were used: rabbit monoclonal anti-TMEM16A [SP31] (ab64085, Abcam) at 1:200, mouse IgG1 anti-CFTR (ab570, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics) at 1:250, mouse polyclonal anti-SLC26A4 (H00005172-A01, Abnova) at 1:200, rabbit polyclonal anti-SLC12A2 (HPA020130, Sigma-Aldrich) at 1:1000, rabbit monoclonal anti-CA2 [EPR5195] (ab124687, Abcam) at 1:500, mouse IgG1 anti-MUC5AC (NCL-HGM-45M1, Novocastra) at 1:100, mouse IgG2B anti-acetylated tubulin (7451, Sigma Aldrich) at 1:300, rabbit polyclonal anti-ATP12A (HPA039526, Sigma-Aldrich) at 1:400.

Techniques: Microscopy, Expressing, Staining

Journal: Scientific Reports

Article Title: Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

doi: 10.1038/srep36016

Figure Lengend Snippet: For simplicity, the cartoon shows all channels and transporters within the same cell although some components (e.g. CFTR and TMEM16A) are localized in separate cell types. The NKCC1 transporter (SLC12A2) promotes the intracellular accumulation of Cl − that is then secreted through TMEM16A and CFTR Cl − channels. Bicarbonate is accumulated inside the cell by means of basolateral transporters and by conversion from CO 2 . Pendrin then mediates the exchange of extracellular Cl − with intracellular HCO 3 − . The apical membrane also contains the ATP12A K + /H + pump and possibly a K + channel. Secretion of K + could be the mechanism controlling the acidification of apical fluid by ATP12A.

Article Snippet: The following primary antibodies and dilutions were used: rabbit monoclonal anti-TMEM16A [SP31] (ab64085, Abcam) at 1:200, mouse IgG1 anti-CFTR (ab570, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics) at 1:250, mouse polyclonal anti-SLC26A4 (H00005172-A01, Abnova) at 1:200, rabbit polyclonal anti-SLC12A2 (HPA020130, Sigma-Aldrich) at 1:1000, rabbit monoclonal anti-CA2 [EPR5195] (ab124687, Abcam) at 1:500, mouse IgG1 anti-MUC5AC (NCL-HGM-45M1, Novocastra) at 1:100, mouse IgG2B anti-acetylated tubulin (7451, Sigma Aldrich) at 1:300, rabbit polyclonal anti-ATP12A (HPA039526, Sigma-Aldrich) at 1:400.

Techniques:

Journal: Neural Regeneration Research

Article Title: Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

doi: 10.4103/1673-5374.268904

Figure Lengend Snippet: Immunoreactivity of NKCC1 and KCC2 in DRG and hippocampal CA1 neurons. (A) Positive control for NKCC1 (acinar cells of the outer secreting portion of rat pancreatic tissue). (B) DRG neurons of rat (NKCC1-positive). (C–E) DRG neurons of CCI models on postoperative days 7, 14, and 21 (NKCC1-positive). (F) Positive control for KCC2 (CA1 neurons in the rat hippocampus). (G) DRG neurons of rats (KCC2-negative, brownish yellow represents NKCC1expression). (H–J) DRG neurons of CCI models on postoperative days 7, 14, and 21 (KCC2-negative) (original magnification, 400×). Black arrows indicate immunohistochemically positive cells. Brownish yellow indicates positive expression of NKCC1 or KCC2. No brownish yellow indicates negative expression of KCC1 or KCC2. Scale bar: 50 µm. (K, L) Histogram of the grayscale values of NKCC1 and KCC2 immunohistochemically positive cells. * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group. Data are expressed as the mean ± SEM ( n = 6; one-way analysis of variance followed by Tukey’s post hoc test). CCI: Chronic constriction injury; DRG: dorsal root ganglion; KCC2: K + -Cl – -cotransporter; NKCC1: Na + -K + -2Cl – cotransporter.

Article Snippet: Membranes were incubated with the rabbit anti-NKCC1 polyclonal antibody (1:1000; Cell Signaling Company, Danfoss Town, Boston, MA, USA), rat anti-KCC2 monoclonal antibody (1:1000; Abcam, Cambridge, UK), or rabbit anti-GAPDH (1:1000) at 4°C overnight, and with goat anti-rabbit lgG (1:10,000; Sugisuke Bridge, Beijing, China) or goat anti-rat lgG (1:10,000; Sugisuke Bridge) at room temperature.

Techniques: Positive Control, Expressing, Control

Journal: Neural Regeneration Research

Article Title: Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

doi: 10.4103/1673-5374.268904

Figure Lengend Snippet: NKCC1-immunoreactive cell count in dorsal root ganglion neurons. Compared with the control group ( n + = 41), the number of medium-sized cells on postoperative days 7 ( n + = 87), 14 ( n + = 81), and 21 ( n + = 83) significantly increased ( n + = 292, n – = 3708). Compared with the control group ( n + = 22), the number of small cells on postoperative day 14 ( n + = 58) significantly increased ( n + = 80, n – = 1920). ** P < 0.01, vs . control group (0 day). Data are expressed as the mean ± SEM (one-way analysis of variance followed by Tukey’s post hoc test). NKCC1: Na + -K + -2Cl – cotransporter. n + : positive cells; n – : negative cells.

Article Snippet: Membranes were incubated with the rabbit anti-NKCC1 polyclonal antibody (1:1000; Cell Signaling Company, Danfoss Town, Boston, MA, USA), rat anti-KCC2 monoclonal antibody (1:1000; Abcam, Cambridge, UK), or rabbit anti-GAPDH (1:1000) at 4°C overnight, and with goat anti-rabbit lgG (1:10,000; Sugisuke Bridge, Beijing, China) or goat anti-rat lgG (1:10,000; Sugisuke Bridge) at room temperature.

Techniques: Cell Counting, Control

Journal: Neural Regeneration Research

Article Title: Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

doi: 10.4103/1673-5374.268904

Figure Lengend Snippet: Protein expression levels of NKCC1 and KCC2 in DRG neurons. (A) Positive control group (cortex cells) of NKCC1 compared with the DRG neurons group. (B) Positive control group (cortex cells) of KCC2 compared with the DRG neurons group. * P < 0.05, ** P < 0.01, vs . DRG group. Data are expressed as the mean ± SEM ( n = 6; independent-sample t -test). NKCC1: Na + -K + -2Cl – cotransporter; KCC2: K + -Cl – -cotransporter; DRG: dorsal root ganglion.

Article Snippet: Membranes were incubated with the rabbit anti-NKCC1 polyclonal antibody (1:1000; Cell Signaling Company, Danfoss Town, Boston, MA, USA), rat anti-KCC2 monoclonal antibody (1:1000; Abcam, Cambridge, UK), or rabbit anti-GAPDH (1:1000) at 4°C overnight, and with goat anti-rabbit lgG (1:10,000; Sugisuke Bridge, Beijing, China) or goat anti-rat lgG (1:10,000; Sugisuke Bridge) at room temperature.

Techniques: Expressing, Positive Control

Journal: Neural Regeneration Research

Article Title: Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

doi: 10.4103/1673-5374.268904

Figure Lengend Snippet: NKCC1 protein expression in dorsal root ganglion neurons of chronic constriction injury models after 7, 14, and 21 days. Surgery side (ipsi) on postoperative day 14 compared with the control group. ** P < 0.01, vs . control group. Data are expressed as the mean ± SEM ( n = 6; one-way analysis of variance followed by Tukey’s post hoc test). NKCC1: Na + -K + -2Cl – cotransporter; ipsi: ipsilateral.

Article Snippet: Membranes were incubated with the rabbit anti-NKCC1 polyclonal antibody (1:1000; Cell Signaling Company, Danfoss Town, Boston, MA, USA), rat anti-KCC2 monoclonal antibody (1:1000; Abcam, Cambridge, UK), or rabbit anti-GAPDH (1:1000) at 4°C overnight, and with goat anti-rabbit lgG (1:10,000; Sugisuke Bridge, Beijing, China) or goat anti-rat lgG (1:10,000; Sugisuke Bridge) at room temperature.

Techniques: Expressing, Control

Journal: Neural Regeneration Research

Article Title: Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

doi: 10.4103/1673-5374.268904

Figure Lengend Snippet: Hypothetical mechanism by which NKCC1 leads to increased excitability of DRG neurons and consequent neuropathic pain. ① In the central nerve terminal of the DRG neurons, the concentration gradient causes Cl – outflow, and GABA mediates primary afferent depolarization (Carlton, 2014). ② The intensity of the primary afferent depolarization is affected by the concentration gradient of Cl – . The higher the Cl – concentration in DRG neurons, the stronger the primary afferent depolarization. Once primary afferent depolarization reaches the threshold of the nerve endings, it induces dorsal root reflex responses (Willis, 1999). ③ Increased expression of NKCC1 in neuropathic pain leads to an influx of Cl- into DRG neurons and an enhanced concentration gradient of Cl – . Primary afferent depolarization is enhanced and the dorsal root reflex is more easily produced. ④ Action potentials caused by the dorsal root reflex can be transmitted to other central nerve terminals of DRG neurons. ⑤ Action potentials travel along fibers to the central nerve terminals of the DRG and then to the neurons in the dorsal spinal cord. NKCC1: Na + -K + -2Cl – cotransporter; DRG: dorsal root ganglion.

Article Snippet: Membranes were incubated with the rabbit anti-NKCC1 polyclonal antibody (1:1000; Cell Signaling Company, Danfoss Town, Boston, MA, USA), rat anti-KCC2 monoclonal antibody (1:1000; Abcam, Cambridge, UK), or rabbit anti-GAPDH (1:1000) at 4°C overnight, and with goat anti-rabbit lgG (1:10,000; Sugisuke Bridge, Beijing, China) or goat anti-rat lgG (1:10,000; Sugisuke Bridge) at room temperature.

Techniques: Concentration Assay, Expressing, Produced

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: American Journal of Physiology - Cell Physiology

Article Title: TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens

doi: 10.1152/ajpcell.00391.2019

Figure Lengend Snippet: Forward and reverse primers

Article Snippet: Rabbit polyclonal anti-phospho-NKCC1 antibody (Thr 212 /Thr 217 , cat. no. ABS1004) was purchased from Millipore Sigma (St. Louis, MO).

Techniques: Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens

doi: 10.1152/ajpcell.00391.2019

Figure Lengend Snippet: RT-PCR revealed sodium-potassium/two-chloride cotransporter 1 (NKCC1) mRNA expression in wild-type (WT) and transient receptor potential vanilloid 1 (TRPV1)-knockout lenses and in cultured epithelium (Epi) obtained from WT and TRPV1−/− lenses. NKCC2 mRNA was not detected. Lanes are identified as follows: Kid, mouse kidney, probed as a positive control; WT, wild type; V1−/−, TRPV1 knockout; −ve, negative control; M, base pair (bp) markers.

Article Snippet: Rabbit polyclonal anti-phospho-NKCC1 antibody (Thr 212 /Thr 217 , cat. no. ABS1004) was purchased from Millipore Sigma (St. Louis, MO).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Knock-Out, Cell Culture, Positive Control, Negative Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens

doi: 10.1152/ajpcell.00391.2019

Figure Lengend Snippet: The influence of capsaicin (1 µM) on sodium-potassium/two-chloride cotransporter 1 (NKCC1) phosphorylation in epithelial cells cultured from wild-type (WT) and transient receptor potential vanilloid 1 (TRPV1)-knockout lenses. A and B: representative Western blots showing phosphorylated NKCC1 (p-NKCC1) and β-actin (loading control) determined in WT (A) and TRPV1−/− (B) cells that were exposed to capsaicin for 1–15 min. Box plots on the right each show phospho-NKCC1 (p-NKCC1) band density (means ± SE) determined by densitometric analysis of 3 independent experiments. **Significant difference from control (P < 0.01). C: Western blot showing p-NKCC1 and β-actin (loading control) determined in 4 independent groups of WT cells at time 0 and following exposure to capsaicin for 2 min. Box plot on the right shows p-NKCC1 band density (means ± SE). ***Significant difference from control (P < 0.001). Underneath is the result of a Western blot carried out after the anti-p-NKCC1 antibody was preabsorbed (blocked) with nonphosphorylated NKCC1 antigenic peptide. The result shows increased p-NKCC1 band density in WT cells exposed to capsaicin for 2 min.

Article Snippet: Rabbit polyclonal anti-phospho-NKCC1 antibody (Thr 212 /Thr 217 , cat. no. ABS1004) was purchased from Millipore Sigma (St. Louis, MO).

Techniques: Phospho-proteomics, Cell Culture, Knock-Out, Western Blot, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens

doi: 10.1152/ajpcell.00391.2019

Figure Lengend Snippet: The increase in rubidium ion (Rb+) uptake in wild-type (WT) cells exposed to capsaicin (1 µM) was prevented by A889425 (1 µM), a TRPV1 antagonist (A), and bumetanide, a sodium-potassium/two-chloride cotransporter (NKCC1) inhibitor (B). Added alone, bumetanide reduced Rb+ uptake. The increase in Rb+ uptake in capsaicin-treated cells was prevented by 10 nM bumetanide as well as 10 μM bumetanide. Data are the mean ± SE of results from 6 to 12 independent experiments. *P < 0.05 and ***P < 0.001, significant differences from control; ###P < 0.001, significant difference from the capsaicin-treated group.

Article Snippet: Rabbit polyclonal anti-phospho-NKCC1 antibody (Thr 212 /Thr 217 , cat. no. ABS1004) was purchased from Millipore Sigma (St. Louis, MO).

Techniques: Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens

doi: 10.1152/ajpcell.00391.2019

Figure Lengend Snippet: Schematic diagram showing 2 arms of the short-term feedback control mechanism that regulates surface cell intracellular hydrostatic pressure. The mechanism is based on the present study as well as previous studies by ourselves and others. The arm that responds to a hyperosmotic stimulus includes a role for transient receptor potential vanilloid 1 (TRPV1) (29, 41), ERK1/2 (29), phosphatidylinositol 3-kinase (PI3K)/Akt (23, 38), WNK-SPAK/OSR1 (1, 2, 17, 35, 41), and sodium-potassium/two-chloride cotransporter 1 (NKCC1) activation (14, 41). The sequence of ERK1/2 leading to PI3K/Akt activation in the scheme is hypothesized. The arm that responds to a hypoosmotic stimulus includes a role for TRPV4 (15, 40, 43), hemichannel opening (Hemi Ch Open), ATP release (40, 43), P2Y receptor, and Src kinase (SFK) activation (28, 40, 43), leading to stimulation of sodium-potassium adenosine triphosphatase (Na,K-ATPase) activity (42, 43). The scheme is a modification of an earlier model (23).

Article Snippet: Rabbit polyclonal anti-phospho-NKCC1 antibody (Thr 212 /Thr 217 , cat. no. ABS1004) was purchased from Millipore Sigma (St. Louis, MO).

Techniques: Control, Activation Assay, Sequencing, Activity Assay, Modification