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: bioRxiv

Article Title: SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

doi: 10.1101/2021.04.27.441695

Figure Lengend Snippet: ( A ) SARS-Cov-2 infection for 90 min (10 5 viral particles exposed apically using enteroid monolayers from 3 different donors) and studied at 48 h reduced mRNA for ACE2, NHE3, and DRA, but not CFTR, Results are means ±sem. *,n=4, p<0.05; ( B , C ) Virus alone reduced total NHE3 expression and the percent of total NHE3 in the apical domain. B , untreated control. C , monolayer infected with 10 5 virus particles for 90 min and studied 2 days later. Representative confocal XY images at level of the BB (i) and XZ cross sections (ii). NHE3 = green; F-actin = red, nuclei = blue. Scale bar = 20µm. SARS-CoV-2 infection decreased total NHE3 fluorescence intensity measured in projections of at least 100 XZ frames in each of two independent experiments from 8879.1 ±1852.8 in control monolayers to 3092.1 ± 1096.2 (p=0.036); SARS-CoV-2 infection decreased the BB NHE3 fluorescence also measured in at least100 XZ frames from 25410.5 ± 5466.7 in control monolayers to 4823.8 ± 4284.8 (p=0.038) in virus infection monolayers.

Article Snippet: Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).

Techniques: Infection, Virus, Expressing, Fluorescence

Journal: bioRxiv

Article Title: SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

doi: 10.1101/2021.04.27.441695

Figure Lengend Snippet: Human ileal enteroid monolayers were exposed apically to VLPs with either S-2PP or S-D614G forms of Spike protein of SARS-CoV-2 in the presence of IL-6/IL-8 (48h, 50 ng/ml). At 48 hours post exposure to VLPs at times 0 and 24 h, total RNA was isolated and mRNA expression of ACE2, NHE3, DRA, and CFTR was measured by qRT-PCR. Red dash = normalization to empty exosome controls. Results are mean ± sem. n=3.

Article Snippet: Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).

Techniques: Isolation, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-COV-2 induced Diarrhea is inflammatory, Ca 2+ Dependent and involves activation of calcium activated Cl channels

doi: 10.1101/2021.04.27.441695

Figure Lengend Snippet: Modeling the molecular mechanisms of SARS-Cov-2 induced diarrhea using a functional interactome created from a small number of proteins that are known to be modulated by the viral infection of human intestinal epithelium ( https://string-db.org/ ). This analysis is based on the most inclusive gene ontology transport pathway (GO:0006810; false discovery rate (FDR) = 2.30e-07); magenta) and includes 21 functionally and physically interacting proteins that are potentially involved in virus-induced signaling that triggers the Isc response in human intestinal epithelium. This model indicates that NHE3 plays a central role in signal transduction between GO:0070102 - the interleukin-6-mediated signaling pathway (red; false discovery rate (FDR)= 4.08e-0) and GO:1902476 - chloride secretion (green, FDR = 5.96e-0). This cross-talk between interleukins and Cl secretion might be mediated by PAT-1 in a CFTR-independent or -dependent manner, leading to ANO1 activation. NHE3 is also crucial for the signal transduction from virus receptor ACE2 via intracellular calcium stores to a membrane Cl/bicarbonate transporter and basolateral voltage-gated potassium channel complex (yellow - CL:8927; FDR = 5.91e-07). The IL-6 functional network (red) together with IL-8 (CXCL8) influences the functions of the SARS-Cov-2 receptor complex and membrane transporters (magenta) which cause intestinal anion secretion via CaCC and via the Ca2+ signaling cascade (blue) that affects the activity of Ca 2+ - or cAMP-mediated basolateral K+ channels (yellow). Following string colors that connect the proteins represent the interactions based on: turquoise - from curated databases; magenta – experimentally determined; green – gene neighborhood; blue – gene co-occurrence; light green – text mining; black – co-expression; light purple – protein homology.

Article Snippet: Forskolin, clotrimazole, BAPTA-AM (Sigma); IL-6, IL-8 (PepProtech); dextran Alexa Fluor 488 (10kDa) and Alexa Fluor 568 (70kDa) (Invitrogen); anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat.# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat # 3525); anti-NHE3 antibodies (Novusbio, Cat# NBP1-82574); anti-ACE2 – monoclonal mouse IgG2A (R&D systems catalog # MAB933); Phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen; Vero E6 cells (ATCC® CRL-1586™).

Techniques: Functional Assay, Infection, Virus, Transduction, Activation Assay, Membrane, Activity Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: COVID-19 Diarrhea is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines

doi: 10.1016/j.jcmgh.2024.101383

Figure Lengend Snippet: Live SARS-CoV-2 reduces NHE3 and DRA but not CFTR mRNAs and NHE3 and DRA but not CFTR protein expression. ( A ) Effect of SARS-CoV-2 infection on the mRNA expression of ACE2, NHE3, DRA, and CFTR human colonoid monolayers. Live SARS-Cov-2 virus was apically exposed for 90 minutes (10 6 PFU/ml) to proximal colonoid monolayers from 3 different donors and studied at 48 hours. There was reduced mRNAs for ACE2, NHE3, and DRA, but not CFTR. Results are means ± SEM; n = 3–5. Normalization was to S18 ribosomal protein mRNA. ( B ) Effect of SARS-CoV-2 infection on the protein expression of NHE3, DRA, and CFTR in human ileal enteroid monolayers studied as in ( A ) above. Results are means ± SEM. In ( B ), data shown as ratio of paired virus/control monolayers due to wide variation among NHE3, DRA, and CFTR protein/GAPDH ratios. n = 3–5 monolayers. P values are comparison with untreated control enteroids; paired t -tests.

Article Snippet: Anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat# 3525); anti-NHE3 antibodies (Novus Biologicals, cat# NBP1- 82574); anti-ACE2 – monoclonal mouse IgG2A (R&D Systems, cat# MAB933); anti-GAPDH antibodies (Sigma, cat# G8795); phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen.

Techniques: Expressing, Infection, Virus, Control, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: COVID-19 Diarrhea is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines

doi: 10.1016/j.jcmgh.2024.101383

Figure Lengend Snippet: SARS-CoV-2 VLPs directly bind and stimulate cytokine/chemokine secretion in human ileal enteroid monolayers and reduce mRNAs for ACE2, NHE3, DRA, and CFTR. ( A ) IF confocal microscopy (XZ plane shown) demonstrating binding of 10 6 particles/ml VLPs were added at times 0 and 24 hours, and monolayers fixed at 48 hours. Exo = empty exosomes; green = anti-rabbit polyclonal antibodies to SARS-COV-2 spike protein; white = phalloidin (actin); blue = Hoechst 33342 (nuclei). Scale bar, 10 um. Similar results of experiments repeated twice. ( B ) Differentiated human ileal enteroid monolayers were apically exposed to empty exosomes (Exo) or 10 6 particles/ml SARS-CoV-2 VLPs, added as in ( A ) at times 0 and 24 hours and sampled at 48 hoours. BL media were collected and analyzed for secreted cytokines/chemokines by multiplex enzyme-linked immunosorbent assay (ELISA). ( C ) VLPs expressing the 4 SARS-CoV-2 structural proteins, including Spike D614G, were exposed apically to ileal enteroid monolayers as in ( B ) and studied at 48 hours, with IL-6 and IL-8 (50 ng/ml each) on the BL surface. Effects on mRNAs demonstrated that, compared with exosomes similarly exposed, there was significant reduction in ACE2, NHE3, DRA, and CFTR. Data shown are normalized to effect of empty exosome controls set as 1.0 for each experiment (horizontal line). Results are means ± SEM. n = 3.

Article Snippet: Anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat# 3525); anti-NHE3 antibodies (Novus Biologicals, cat# NBP1- 82574); anti-ACE2 – monoclonal mouse IgG2A (R&D Systems, cat# MAB933); anti-GAPDH antibodies (Sigma, cat# G8795); phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen.

Techniques: Confocal Microscopy, Binding Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: COVID-19 Diarrhea is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines

doi: 10.1016/j.jcmgh.2024.101383

Figure Lengend Snippet: Irradiated virus is taken up in human colonic enteroid monolayers but does not alter enterocyte cytokine secretion or mRNAs of NHE3, DRA, CFTR, TMEM16F, and TMEM16, whereas IL-6 plus IL-8 reduce mRNAs of NHE3 and DRA. ( A ) 10 6 viral particles/ml of irradiated virus were exposed apically to differentiated human enteroid monolayers for various times and intracellular virus identified by IB using anti-Spike antibodies. Representative study, repeated 4 times, is shown. Uptaken Spike is present at 2 and 4 but not 24 hours after exposure. ( B ) Irradiated virus exposure for 48 hours did not alter cytokines/chemokine secretion from differentiated ileal enteroid monolyes. 10 6 PFU/ml irradiated virus was added apically at times 0 and 24 hours to ileal enteroid monolayers and BL media sampled at 48 hours for cytokines and chemokine assays. In parallel, enteroids that were not treated were used as controls. Data shown set the untreated controls as 1 for each experiment. Two separate ileal enteroid lines were studied. Results are means ± SEM; n = 6. ( C ) IL-6/IL-8 but not irradiated virus decreased mRNAs of NHE3 and DRA but not CFTR, TMEM16A, or TMEM16F. Differentiated ileal enteroid monolayers from 3 separate ileal enteroid cultures were exposed on the apical surface to 10 6 PFU/ml irradiated virus, the combination of irradiated virus with IL-6/IL-8 (50 nl/ml each) on the BL surface, or the IL-6/IL-8 alone. Irradiated virus was added at times 0 and 24 hours and IL-6/IL-8 at time 0 and all sampled at 48 hours for mRNA determinations. Results are means ± SEM; n = 4–9. ANOVA used to calculate P values shown above bars. Bracketed P values compare IL-6/IL-8 effect to combination of irradiated virus plus IL-6/IL-8. ( D ) Irradiated virus exposure for 48 hours did not alter NHE3 protein expression ( left ), but IL-6 plus IL-8 for this time reduced NHE3 ( right ). Three × 10 6 /ml irradiated virus was exposed apically at times 0 and 24 hours to ileal enteroid monolayers to IL-6/IL-8 (50 ng/ml each) added at time 0 to the BL surface, and 48 after initial exposure, the entroids were lysed for IB or fixed for IF study of NHE3. Irradiated virus alone did not alter NHE3 protein expression by IB ( left ). Right : representative confocal XY images at the level of the the BB ( Ei, Eiii ) and XZ sections ( Eii, Eiv ). Ei, Eii, untreated controls; Eiii, Eiv, irradiated virus. NHE3, green ; F-actin, red ; nuclei, blue . IL-6/IL-8 greatly reduced total NHE3 protein expression. Scale bar, 20 um. Experiment on left was done once, and those on right were repeated 3 times.

Article Snippet: Anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat# 3525); anti-NHE3 antibodies (Novus Biologicals, cat# NBP1- 82574); anti-ACE2 – monoclonal mouse IgG2A (R&D Systems, cat# MAB933); anti-GAPDH antibodies (Sigma, cat# G8795); phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen.

Techniques: Irradiation, Virus, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: COVID-19 Diarrhea is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines

doi: 10.1016/j.jcmgh.2024.101383

Figure Lengend Snippet: Gene-specific Primers for qRT-PCR

Article Snippet: Anti-dsRNA monoclonal antibodies (clone rJ2; Millipore, cat# MABE1134); anti-spike rabbit polyclonal antibodies (ProSci, cat# 3525); anti-NHE3 antibodies (Novus Biologicals, cat# NBP1- 82574); anti-ACE2 – monoclonal mouse IgG2A (R&D Systems, cat# MAB933); anti-GAPDH antibodies (Sigma, cat# G8795); phalloidin and secondary fluorescent AlexaFluor antibodies from Invitrogen.

Techniques:

Journal: Frontiers in Microbiology

Article Title: EGFR as a Negative Regulatory Protein Adjusts the Activity and Mobility of NHE3 in the Cell Membrane of IPEC-J2 Cells With TGEV Infection

doi: 10.3389/fmicb.2018.02734

Figure Lengend Snippet: The effect of TGEV on Na + concentration and the relative expression of NHE3 in IPEC-J2 cells. (A,B) Intra and extracellular Na + concentration in cells infected with TGEV at 0, 48, and 72 h post infection. (C) NHE3 mRNA expression levels were detected using qRT-PCR and normalized by the β-actin mRNA level. (D) NHE3 protein levels were analyzed by Western blotting. (E) Grayscale analysis of NHE3 relative abundance changes. ∗ 0.01< p < 0.05, ∗∗ p < 0.01.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit polyclonal anti-NHE3 (Affinity), rabbit polyclonal anti-EGFR (Biorbyt), rabbit polyclonal anti-phospho-EGF (Tyr1068) (Cell Signaling), rabbit polyclonal anti-phospho-ERK1/2 (Thr202+Tyr204) (Bioss), rabbit polyclonal anti-β-tubulin (Proteintech), with goat anti-rabbit IgG (H+L) antibodies (Sangon Biotech) as the secondary antibody.

Techniques: Concentration Assay, Expressing, Infection, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Microbiology

Article Title: EGFR as a Negative Regulatory Protein Adjusts the Activity and Mobility of NHE3 in the Cell Membrane of IPEC-J2 Cells With TGEV Infection

doi: 10.3389/fmicb.2018.02734

Figure Lengend Snippet: TGEV regulated the inhibition of NHE3 via the EGFR/ERK pathway. (A) IPEC-J2 cells in corresponding groups were treated with AG1478, protein levels of NHE3, p-EGFR, EGFR, p-ERK, and ERK in TGEV-infected cells or uninfected cells were analyzed by Western blotting using specific antibodies. (B–D) Grayscale analysis of the changes in NHE3, p-EGFR/EGFR, and p-ERK/ERK levels, as analyzed using the spass software. (E,F) Knockdown of EGFR expression in IPEC-J2 cells by short hairpin shEGFR lentivirus was decreased by 57–66% in shEGFR-infected cells compared with control cells. (G) IPEC-J2 cells in corresponding groups were treated with EGFR-specific shRNA, expression levels of p-EGFR, EGFR, p-ERK, ERK, and NHE3 proteins were evaluated by Western blotting analysis. (H–J) Grayscale analysis of the changes in p-EGFR/EGFR, p-ERK/ERK, and NHE3 levels, as analyzed using the spass software. Each experiment was performed in triplicate. ∗ 0.01< p < 0.05, ∗∗ p < 0.01.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit polyclonal anti-NHE3 (Affinity), rabbit polyclonal anti-EGFR (Biorbyt), rabbit polyclonal anti-phospho-EGF (Tyr1068) (Cell Signaling), rabbit polyclonal anti-phospho-ERK1/2 (Thr202+Tyr204) (Bioss), rabbit polyclonal anti-β-tubulin (Proteintech), with goat anti-rabbit IgG (H+L) antibodies (Sangon Biotech) as the secondary antibody.

Techniques: Inhibition, Infection, Western Blot, Software, Knockdown, Expressing, Control, shRNA

Journal: Frontiers in Microbiology

Article Title: EGFR as a Negative Regulatory Protein Adjusts the Activity and Mobility of NHE3 in the Cell Membrane of IPEC-J2 Cells With TGEV Infection

doi: 10.3389/fmicb.2018.02734

Figure Lengend Snippet: Fluorescence observation of IPEC-J2 cells transfected with pEGFP-NHE3 (10×). IPEC-J2 cells were transfected with recombinant vector (pEGFP-NHE3) for different times (24, 48, 72, and 120 h), and examined using fluorescence/phase-contrast microscopy.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit polyclonal anti-NHE3 (Affinity), rabbit polyclonal anti-EGFR (Biorbyt), rabbit polyclonal anti-phospho-EGF (Tyr1068) (Cell Signaling), rabbit polyclonal anti-phospho-ERK1/2 (Thr202+Tyr204) (Bioss), rabbit polyclonal anti-β-tubulin (Proteintech), with goat anti-rabbit IgG (H+L) antibodies (Sangon Biotech) as the secondary antibody.

Techniques: Fluorescence, Transfection, Recombinant, Plasmid Preparation, Microscopy

Journal: Frontiers in Microbiology

Article Title: EGFR as a Negative Regulatory Protein Adjusts the Activity and Mobility of NHE3 in the Cell Membrane of IPEC-J2 Cells With TGEV Infection

doi: 10.3389/fmicb.2018.02734

Figure Lengend Snippet: Fluorescence recovery rate on the surface of IPEC-J2 cell (2 mW, 63×/1.4 NA). FRAP recovery curves of pEGFP-NHE3 in different groups are shown. Error bars represent the mean ± SD.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit polyclonal anti-NHE3 (Affinity), rabbit polyclonal anti-EGFR (Biorbyt), rabbit polyclonal anti-phospho-EGF (Tyr1068) (Cell Signaling), rabbit polyclonal anti-phospho-ERK1/2 (Thr202+Tyr204) (Bioss), rabbit polyclonal anti-β-tubulin (Proteintech), with goat anti-rabbit IgG (H+L) antibodies (Sangon Biotech) as the secondary antibody.

Techniques: Fluorescence

Journal: Frontiers in Microbiology

Article Title: EGFR as a Negative Regulatory Protein Adjusts the Activity and Mobility of NHE3 in the Cell Membrane of IPEC-J2 Cells With TGEV Infection

doi: 10.3389/fmicb.2018.02734

Figure Lengend Snippet: Analysis of NHE3 mobile fraction (2 mW, 63×/1.4 NA). Each test was replicated three times. FRAP data from eight different cells in each group were used for the calculation and analysis. ∗ 0.01< p < 0.05, ∗∗ p < 0.01.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit polyclonal anti-NHE3 (Affinity), rabbit polyclonal anti-EGFR (Biorbyt), rabbit polyclonal anti-phospho-EGF (Tyr1068) (Cell Signaling), rabbit polyclonal anti-phospho-ERK1/2 (Thr202+Tyr204) (Bioss), rabbit polyclonal anti-β-tubulin (Proteintech), with goat anti-rabbit IgG (H+L) antibodies (Sangon Biotech) as the secondary antibody.

Techniques:

Journal: Frontiers in Microbiology

Article Title: EGFR as a Negative Regulatory Protein Adjusts the Activity and Mobility of NHE3 in the Cell Membrane of IPEC-J2 Cells With TGEV Infection

doi: 10.3389/fmicb.2018.02734

Figure Lengend Snippet: The mechanism of the regulation of NHE3 activity by the EGFR/ERK signaling pathway in intestinal epidermal cells after TGEV infection.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit polyclonal anti-NHE3 (Affinity), rabbit polyclonal anti-EGFR (Biorbyt), rabbit polyclonal anti-phospho-EGF (Tyr1068) (Cell Signaling), rabbit polyclonal anti-phospho-ERK1/2 (Thr202+Tyr204) (Bioss), rabbit polyclonal anti-β-tubulin (Proteintech), with goat anti-rabbit IgG (H+L) antibodies (Sangon Biotech) as the secondary antibody.

Techniques: Activity Assay, Infection