Journal: Frontiers in Immunology

Article Title: Promoting intestinal antimicrobial defense and microbiome symbiosis contributes to IL-22-mediated protection against alcoholic hepatitis in mice

doi: 10.3389/fimmu.2023.1289356

Figure Lengend Snippet: Primer sequences used for qPCR analysis.

Article Snippet: Cryostat sections of mouse ileum or liver tissues were incubated with anti-IL-22 (Genetex, Irvine, CA; Cat. #GTX18498), anti-F4/80 (BD Biosciences, Franklin Lakes, NJ; Cat. #565409), anti-MPO (Lifespan Biosciences, Seattle, WA; Cat. #LS-B6699), anti-DEFA5 (Elabscience, Houston, TX; ESAP13305), anti-ZO-1 (Millipore, Burlington, MA; Cat. #MABT11), anti-Ki67 antibody (Millipore; Cat. #AB9260), or anti-sodium hydrogen antiporter 3 (NHE3) antibody (Novus Biologicals, Centennial, CO; Cat. # NBP1-82574) followed by Alexa Fluor 594-conjugated IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Promoting intestinal antimicrobial defense and microbiome symbiosis contributes to IL-22-mediated protection against alcoholic hepatitis in mice

doi: 10.3389/fimmu.2023.1289356

Figure Lengend Snippet: IL-22 stimulates IEC differentiation in a STAT3-depedent manner in mice and cultured organoids. WT mice were subjected to an 8-wk alcohol feeding, and IL-22 was i.p. injected at 1 mg/kg for the last 2 wk of feeding. (A) IF staining of ileal Ki67 (red) and nuclei (blue). Scale bar, 50 μm. (B) IF staining of ileal NHE3 (red) and nuclei (blue). Scale bar, 50 μm. (C) Organoid forming efficiency assessed by bright field imaging. Scale bar, 100 μm. Small intestinal organoids isolated from Stat3 fl/fl and Stat3 IEC-/- mice were treated with 100 ng/ml IL-22 for 10 h. (D) The mRNA levels of Nhe3 in organoids isolated from Stat3 fl/fl and Stat3 IEC-/- mice after IL-22 treatment. *** P < 0.001. (E) IF staining of NHE3 (red) in organoids after IL-22 treatment. Nuclei were stained by DAPI (blue). Scale bar, 20 μm.

Article Snippet: Cryostat sections of mouse ileum or liver tissues were incubated with anti-IL-22 (Genetex, Irvine, CA; Cat. #GTX18498), anti-F4/80 (BD Biosciences, Franklin Lakes, NJ; Cat. #565409), anti-MPO (Lifespan Biosciences, Seattle, WA; Cat. #LS-B6699), anti-DEFA5 (Elabscience, Houston, TX; ESAP13305), anti-ZO-1 (Millipore, Burlington, MA; Cat. #MABT11), anti-Ki67 antibody (Millipore; Cat. #AB9260), or anti-sodium hydrogen antiporter 3 (NHE3) antibody (Novus Biologicals, Centennial, CO; Cat. # NBP1-82574) followed by Alexa Fluor 594-conjugated IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Cell Culture, Injection, Staining, Imaging, Isolation

Journal: Frontiers in Immunology

Article Title: Promoting intestinal antimicrobial defense and microbiome symbiosis contributes to IL-22-mediated protection against alcoholic hepatitis in mice

doi: 10.3389/fimmu.2023.1289356

Figure Lengend Snippet: Schematic figure summarizing the major findings of the present study (created with BioRender.com). AMP, antimicrobial peptide; IEC, intestinal epithelial cells; NHE3, sodium-hydrogen exchanger 3; PAMP, pathogen-associated molecular pattern; STAT3, signal transducer and activator of transcription 3.

Article Snippet: Cryostat sections of mouse ileum or liver tissues were incubated with anti-IL-22 (Genetex, Irvine, CA; Cat. #GTX18498), anti-F4/80 (BD Biosciences, Franklin Lakes, NJ; Cat. #565409), anti-MPO (Lifespan Biosciences, Seattle, WA; Cat. #LS-B6699), anti-DEFA5 (Elabscience, Houston, TX; ESAP13305), anti-ZO-1 (Millipore, Burlington, MA; Cat. #MABT11), anti-Ki67 antibody (Millipore; Cat. #AB9260), or anti-sodium hydrogen antiporter 3 (NHE3) antibody (Novus Biologicals, Centennial, CO; Cat. # NBP1-82574) followed by Alexa Fluor 594-conjugated IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques:

Journal: Biology of Reproduction

Article Title: Relative contribution of clear cells and principal cells to luminal pH in the mouse epididymis †

doi: 10.1095/biolreprod.116.144857

Figure Lengend Snippet: Expression of NHE2 and NHE3 in the mouse cauda epididymidis. (A) Double labeling for NHE3 (green) and V-ATPase (red) showed strong expression of NHE3 in the apical membrane of PCs (arrows). No expression was detected in CCs, labeled for the V-ATPase. Rows of CCs are sometimes observed in the mouse cauda epididymidis (arrowheads). (B) Double labeling for NHE2 (green) and V-ATPase (red) showed no expression of NHE2 in neither PCs nor CCs, identified by their immunolabeling for V-ATPase (arrowheads). (C) RT-PCR detection of NHE3 mRNA in kidney (K, used as a positive control), testis (T, also used as a positive control), epididymis initial segment (IS), caput (Cp), and cauda (Cd). A strong signal was observed in all samples. M = markers. (D) Western blot detection of NHE3 in total membrane and cytosolic fraction in kidney (K), testis (T), caput (Cp), and cauda (Cd). A strong signal was observed in the membrane fraction and not in the cytosol. (A, B) Scale bars = 20 μm.

Article Snippet: Overnight incubation was performed at 4°C with rabbit anti-NHE3 antibody (Santa Cruz, Santa Cruz, CA, USA, sc-16103-R, 1:1,000 dilution) followed by HRP-conjugated goat anti-rabbit secondary antibody at a dilution of 1:5,000 for 1 h at room temperature.

Techniques: Expressing, Labeling, Membrane, Immunolabeling, Reverse Transcription Polymerase Chain Reaction, Positive Control, Western Blot

Journal: Biology of Reproduction

Article Title: Relative contribution of clear cells and principal cells to luminal pH in the mouse epididymis †

doi: 10.1095/biolreprod.116.144857

Figure Lengend Snippet: Effect of luminal pH and cpt-cAMP on the subcellular localization of NHE3. (A) Cell fractionation WB analysis of NHE3 in plasma membrane and vesicle fractions from the cauda epididymis perfused at pH 6.6 versus pH 7.8. An increase in the amount of NHE3 located in the plasma membrane versus intracellular vesicles. *P < 0.05 versus pH 6.6. (B) Perfusion at pH 7.8 in the presence of cpt-cAMP reduced the ratio of NHE3 present in plasma membrane versus vesicles compared to pH 7.8 alone. *P < 0.05 versus pH 7.8 alone.

Article Snippet: Overnight incubation was performed at 4°C with rabbit anti-NHE3 antibody (Santa Cruz, Santa Cruz, CA, USA, sc-16103-R, 1:1,000 dilution) followed by HRP-conjugated goat anti-rabbit secondary antibody at a dilution of 1:5,000 for 1 h at room temperature.

Techniques: Cell Fractionation, Clinical Proteomics, Membrane

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Western blot and quantification of Na+/H+ exchanger 3 (NHE3) and vacuolar H+-ATPase (V-ATPase) expression of whole kidney (A) and medulla (B) of mice treated with HK + control (Ctrl) and HK + furosemide (Furo). *P < 0.05 vs. HK + Ctrl analyzed with Student’s t-test; n = 4/group.

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Niche-inspired synthetic matrices for epithelial organoid culture

doi: 10.1101/806919

Figure Lengend Snippet: a , Bright-field images of 10-day old human enteroids growing in the synthetic ECM showing diverse phenotypes upon inducing differentiation. Scale bar: 200 μm. b-c , Immunostaining analysis of 10-day old organoids in the synthetic ECM and Matrigel showing proliferative cells (Ki67), Paneth cells (Lyz), Goblet cells (Muc2). Epithelial marker (E-cad), apical marker (NHE3) and basolateral markers (CD44v6, C-IV, and LMN). Scale bar: 500 μm.

Article Snippet: Immunostaining for cell-specific markers was done using the following primary antibodies, diluted in blocking solution; goat anti-E-cadherin (R&D, AF748, 1:200), goat anti-hDPPIV/CD26 (Invitrogen, AF1180, 1:400), rabbit anti-Col IV (Abcam, Ab6586, 1:200), rabbit anti-Lysozyme (Dako, A0099, 1:200), rabbit anti-LMN (Abcam, Ab11575, 1:200), rabbit anti-Ki67, (Abcam, Ab15580, 1:200 or ab16667, 1:100), rabbit anti-NHE3/SCL9A3 (Novus, NBP-82574), mouse anti-Villin (Santa Cruz, SC-58897, 1:50), mouse anti-CD44-v6 (Abcam, Ab78960, 1:200), mouse anti-Muc2 (Santa Cruz, SC515032 1:200), mouse anti-EpCAM (Abcam, ab7504 1:200).

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Niche-inspired synthetic matrices for epithelial organoid culture

doi: 10.1101/806919

Figure Lengend Snippet: a-b , Immunostaining analysis of 10-day old organoids differentiated in the synthetic ECM and Matrigel showing proliferative cells (Ki67), Paneth cells (Lyz), epithelial marker (E-cad), apical markers (NHE3, Villin) and basolateral markers (CD44v6, C-IV, and LMN). Scale bar: 50 μm.

Article Snippet: Immunostaining for cell-specific markers was done using the following primary antibodies, diluted in blocking solution; goat anti-E-cadherin (R&D, AF748, 1:200), goat anti-hDPPIV/CD26 (Invitrogen, AF1180, 1:400), rabbit anti-Col IV (Abcam, Ab6586, 1:200), rabbit anti-Lysozyme (Dako, A0099, 1:200), rabbit anti-LMN (Abcam, Ab11575, 1:200), rabbit anti-Ki67, (Abcam, Ab15580, 1:200 or ab16667, 1:100), rabbit anti-NHE3/SCL9A3 (Novus, NBP-82574), mouse anti-Villin (Santa Cruz, SC-58897, 1:50), mouse anti-CD44-v6 (Abcam, Ab78960, 1:200), mouse anti-Muc2 (Santa Cruz, SC515032 1:200), mouse anti-EpCAM (Abcam, ab7504 1:200).

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Niche-inspired synthetic matrices for epithelial organoid culture

doi: 10.1101/806919

Figure Lengend Snippet: Immunostaining analysis of 10-day old organoids emerging in the synthetic matrix and Matrigel showing proliferative cells (Edu), and correct localization of apical markers (F-actin, NHE3 and DPP4).

Article Snippet: Immunostaining for cell-specific markers was done using the following primary antibodies, diluted in blocking solution; goat anti-E-cadherin (R&D, AF748, 1:200), goat anti-hDPPIV/CD26 (Invitrogen, AF1180, 1:400), rabbit anti-Col IV (Abcam, Ab6586, 1:200), rabbit anti-Lysozyme (Dako, A0099, 1:200), rabbit anti-LMN (Abcam, Ab11575, 1:200), rabbit anti-Ki67, (Abcam, Ab15580, 1:200 or ab16667, 1:100), rabbit anti-NHE3/SCL9A3 (Novus, NBP-82574), mouse anti-Villin (Santa Cruz, SC-58897, 1:50), mouse anti-CD44-v6 (Abcam, Ab78960, 1:200), mouse anti-Muc2 (Santa Cruz, SC515032 1:200), mouse anti-EpCAM (Abcam, ab7504 1:200).

Techniques: Immunostaining

Journal: Nutrients

Article Title: Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na + /K + -ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes

doi: 10.3390/nu10030302

Figure Lengend Snippet: Association of transport proteins with the DRM fraction prepared from jejunal and ileal apical membranes after incubation with resveratrol (300 µM, 30 min) expressed as changes (means ± SD) in the protein/flotillin-1 ratio. The change in protein/flotillin-1 ratio was calculated (the protein/flotillin-1 ratio for resveratrol-treated tissues was set in relation to preparations from control tissues ( n = 6, n = 4 for pNHE3 Ser605 Ileum)).

Article Snippet: Additionally, the following primary antibodies and conditions were used: NHE3: denaturing at RT for 20 min, primary antibodies: rabbit-anti-NHE3 (sc-16103-R, Santa Cruz Biotechnology, Dallas, TX, USA), mouse-anti-NHE3 Ser552 (NB110-81529, Novus Biologicals, Littleton, CO, USA, mouse-anti-NHE3 Ser605 (NB110-74678SS, Novus Biologicals); PEPT1, Na + /K + -ATPase, flotillin-1: denaturing at 95 °C for 5 min, goat-anti-PEPT1 (19917, Santa Cruz), mouse-anti-Na + /K + -ATPase (ALX-804-082, Enzo life Sciences, New York, NY, USA), rabbit-anti-flotillin-1 (3253, Cell Signaling).

Techniques: Incubation, Control