Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The stained cells were resuspended in RNAse inhibitor containing sort buffer (RNAsin, Promega, 200U/μl) and sorted by gating on γδTCR + CD3 + cells directly into a 384-well PCR plate that had been preloaded with 1μl of reverse transcription reaction mix [1X SuperScript VILO Reaction Mix (Thermo Fisher), SuperScript VILO enzyme and 1X0.1% NP40 (Thermo Fisher)] with a sorter (Model SY3200, Sony Biotech Synergy sorter, Sony Biotech, San Jose, CA).

Techniques: Control, Virus, Plasmid Preparation, Recombinant, Cell Stimulation, Protease Inhibitor, Blocking Assay, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Sequencing, Software

Journal: Advanced Functional Materials

Article Title: Tumor‐On‐A‐Chip Model Incorporating Human‐Based Hydrogels for Easy Assessment of Metastatic Tumor Inter‐Heterogeneity

doi: 10.1002/adfm.202315940

Figure Lengend Snippet: Figure 5. Gene and protein biomarkers expression depending on tumor metastatic ability. a) Relative gene expression of VEGFA, COL1A1, MMP2, and MMP9 in the (i) low-metastatic and (ii) high-metastatic models for the different culture conditions, normalized to the static culture. b) Comparison of low- and high-metastatic relative gene expression of (i) VEGFA, (ii) COL1A1, and (iii) MMP2, for each culture condition. c) ELISA quantification of VEGFA protein secretion at days 3, 4, and 7. Statistical significance between different culture conditions, for the same time-point, is represented with a solid line. Unless otherwise stated, time-dependent significance in each culture condition and model is statistically different. d) Confocal images of Ezrin expression (green) in the low-metastatic model, at 7 days of culture. Actin filaments and nuclei are stained in red and blue, respectively. Scale bar: 200 μm. Data are presented as mean ± SD (n ≥3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns: not significant.

Article Snippet: The samples were incubated with primary mouse monoclonal anti-human Ezrin antibody (1:50 in 5% FBS (v/v) in PBS, 3C12, Santa Cruz Biotechnology, USA) at 4 °C for 3 days, followed by PBS washing for 1 h, and then incubated with the secondary antibody goat antimouse Alexa Fluor 488 (1:100 in 5% FBS (v/v) in PBS, Thermo Fisher Scientific, USA) at 4 °C for 3 days.

Techniques: Expressing, Gene Expression, Comparison, Enzyme-linked Immunosorbent Assay, Staining

Journal: eLife

Article Title: Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

doi: 10.7554/eLife.33432

Figure Lengend Snippet: ( A ) Intracellular pH (pHi) of the ligated efferent ductules from WT (n = 9) mice and Adgrg2 -/Y (n = 9) mice were measured by carboxy-SNARF (5 μM), with or without incubation with the CFTR inhibitor CFTRinh-172. ( B ) qRT-PCR analysis of CFTR levels in the efferent ductules of WT (n = 3) or Adgrg2 -/Y (n = 3) mice. ( C ) Co-localization of ADGRG2 (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the male efferent ductules of WT mice. Scale bars, 50 μm. ( D ) Analysis of ADGRG2 and CFTR fluorescence intensities by Pearson’s correlation analysis. The Pearson's correlation coefficient was 0.76. ( E ) Immunofluorescence staining of ADGRG2 (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the efferent ductules of Adgrg2 -/Y mice. Scale bars, 50 μm. ( F ) Co-localization of ADGRG2 (red fluorescence) and ezrin (green fluorescence) in the male efferent ductules of WT mice. Scale bars, 50 μm. ( G ) Analysis of ADGRG2 and ezrin fluorescence intensities by Pearson’s correlation analysis. The Pearson's correlation coefficient was 0.69. ( H ) ADGRG2 was immunoprecipitated with an anti-ADGRG2 antibody from the male efferent ductules of WT mice or Adgrg2 -/Y mice, and co-precipitated CFTR, Gs, Gq, β-arrestin-1, β-arrestin-2 and Gi-1/2/3 levels were examined by using specific corresponding antibodies (CFTR antibody:20738–1-AP, Proteintech). (5A-5B) *p < 0.05, **p < 0.01, ***p < 0.001, Adgrg2 -/Y mice compared with WT mice. #p < 0.05, ##p < 0.01, ###p < 0.001. Treatment with selective inhibitors or stimulators was compared with control vehicles. n.s., no significant difference. At least three independent biological replicates were performed for .

Article Snippet: Antibody , Ezrin antibody(rabbit polyclonal) , Proteintech , RRID: AB_2722561 , .

Techniques: Incubation, Quantitative RT-PCR, Fluorescence, Immunofluorescence, Staining, Immunoprecipitation, Control

Journal: eLife

Article Title: Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

doi: 10.7554/eLife.33432

Figure Lengend Snippet: ( A ) Diameters of the luminal ductules derived from WT (n = 12), Adgrg2 -/Y (n = 12) or Arrb1 -/- (n = 15) mice. ( B ) Diameters of the luminal ductules derived from WT (n = 12), Adgrg2 -/Y (n = 12) or Arrb2 -/- (n = 15) mice. ( C ) Intracellular pH (pHi) of the ligated efferent ductules derived from WT (n = 9), Arrb1 -/- (n = 9) or Arrb2 -/- (n = 9) mice were measured by carboxy-SNARF. ( D ) Co-localization of ADGRG2 (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the male efferent ductules of Arrb2 -/- mice. ( E ) Analysis of ADGRG2 and CFTR fluorescence intensities in Arrb2 -/- mice by Pearson’s correlation analysis. The Pearson's correlation coefficient was 0.62. ( F ) Localization of ADGRG2 (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the male efferent ductules of Arrb1 -/- mice. ( G ) Analysis of ADGRG2 and CFTR fluorescence intensities in Arrb1 -/- mice by Pearson’s correlation analysis. The Pearson's correlation coefficient was −0.15. ( H ) Co-localization of ezrin (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the male efferent ductules of Arrb2 -/- mice. ( I ) Analysis of ezrin and CFTR fluorescence intensities in Arrb2 -/- mice by Pearson’s correlation analysis. The Pearson's correlation coefficient was 0.66. ( J ) Co-localization of ezrin (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the male efferent ductules of Arrb1 -/- mice. ( K ) Analysis of ezrin and CFTR fluorescence intensities in Arrb1 -/- mice by Pearson’s correlation analysis. The Pearson's correlation coefficient was −0.15. ( L ) ADGRG2 was immunoprecipitated by an anti-ADGRG2 antibody in the male efferent ductules of Arrb1 -/- mice or Arrb2 -/- mice, and co-precipitates with CFTR, β-arrestin-1, and β-arrestin-2 were examined by using specific corresponding antibodies (CFTR antibody:20738–1-AP, Proteintech). (9A-C) *p < 0.05, **p < 0.01, ***p < 0.001, Adgrg2 -/Y mice compared with WT mice. #p < 0.05, ##p < 0.01, ###p < 0.001, Arrb1 -/- mice or Arrb2 -/- mice compared with WT mice. ns, no significant difference. At least three independent biological replicates were performed for .

Article Snippet: Antibody , Ezrin antibody(rabbit polyclonal) , Proteintech , RRID: AB_2722561 , .

Techniques: Derivative Assay, Fluorescence, Immunoprecipitation

Journal: eLife

Article Title: Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

doi: 10.7554/eLife.33432

Figure Lengend Snippet: ( A ) Co-localization of Ezrin (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in male efferent ductules of the WT mice and Adgrg2 -/Y mice. Scale bars, 50 μm. Analysis of Ezrin and CFTR fluorescence intensities by Pearson’s correlation. The pearson's correlation coefficient is 0.6 for WT mice and 0.65 for Adgrg2 -/Y mice. ( B ) Bar graph representation and statistical analyses of . ***p < 0.001, Arrb2 -/- lysates or IP protein were compared with Arrb1 -/- lysates or IP protein, respectively. n.s., no significant difference.

Article Snippet: Antibody , Ezrin antibody(rabbit polyclonal) , Proteintech , RRID: AB_2722561 , .

Techniques: Fluorescence

Journal: eLife

Article Title: Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

doi: 10.7554/eLife.33432

Figure Lengend Snippet:

Article Snippet: Antibody , Ezrin antibody(rabbit polyclonal) , Proteintech , RRID: AB_2722561 , .

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reporter Assay

Journal: Cell

Article Title: Serotonin reduction in post-acute sequelae of viral infection

doi: 10.1016/j.cell.2023.09.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Vil1 Taqman assay , Thermo Scientific , Mm00494146_m1.

Techniques: Virus, Clinical Proteomics, Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bicinchoninic Acid Protein Assay, SYBR Green Assay, TaqMan Assay, Software, Imaging, Control, Electron Microscopy, Low Protein Binding, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

doi: 10.1186/s12964-022-00879-3

Figure Lengend Snippet: LPS induced ezrin phosphorylation in a concentration- and time-dependent manner. A549 and HPAEpiC cells were treated with LPS for 3 h at concentrations of 0 µg/mL, 0.1 µg/mL, 1 µg/mL, and 10 µg/mL, respectively. Phosphorylated ezrin protein level was evaluated by western blotting ( a , b ) and flow cytometry ( c , d ). A549 and HPAEpiC cells were treated with LPS (1 µg/mL) for 0 h, 0.5 h, 1 h, 3 h, 6 h and 12 h, respectively. Phosphorylated ezrin protein level was evaluated by western blotting ( e , f ) and flow cytometry ( g , h ). Data are expressed as means ± SD of triplicate samples. * p < 0.05 versus 0 µg/mL group

Article Snippet: For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

doi: 10.1186/s12964-022-00879-3

Figure Lengend Snippet: ROCK mediated LPS-induced ezrin phosphorylation and translocation. A549 and HPAEpiC cells were challenged with LPS (1 µg/mL) in the presence or absence of ROCK siRNA or Y-27632. Total ezrin and p-ezrin protein levels were detected by western blotting using β-actin as an internal reference ( a , b ). Data are expressed as means ± SD of triplicate samples. * p < 0.05 versus control, # p < 0.05 versus LPS. The intracellular localization of p-ezrin in resting and LPS-activated cells was investigated by immunofluorescence. P-ezrin was stained with Alexa Fluor 488-conjugated IgG (green), F-actin was stained with Rhodamine-phalloidin (yellow), and nuclei were stained with DAPI (blue) ( c , d ). Qualitative analysis was performed by confocal microscopy. Scale bar, 10 μm. All experiments were performed in three independent experiments

Article Snippet: For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Control, Immunofluorescence, Staining, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

doi: 10.1186/s12964-022-00879-3

Figure Lengend Snippet: The association between ezrin, MyD88/IRAK1, and Syk. A549 cells were challenged with LPS (1 µg/mL) in the presence or absence of ROCK siRNA or ezrin siRNA. Lysates isolated from A549 cells exposed to LPS (+) or without LPS (−) were immunoprecipitated in the presence of anti-ezrin or control IgG antibody, followed by western blotting with anti-Syk, anti-MyD88, or anti-IRAK1 antibodies, respectively. IP: immunoprecipitation, IB: immunoblotting

Article Snippet: For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.

Techniques: Isolation, Immunoprecipitation, Control, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

doi: 10.1186/s12964-022-00879-3

Figure Lengend Snippet: The suppression of ezrin inhibited LPS-induced activation of p38 and NF-κB. A549 cells were transfected with ezrin siRNA or control siRNA. The expression of ezrin mRNA was detected by qRT-PCR ( a ) and the protein level of ezrin was measured by western blotting ( b ). The effect of ezrin siRNA transfection on IKK, IκBα, and MAPKs activation was detected by western blotting ( c – h ), and NF-κB activation was determined by EMSA (i). Data are expressed as mean ± SD of triplicate samples. * p < 0.05 versus LPS(-) control siRNA group, # p < 0.05 versus LPS(+) control siRNA group

Article Snippet: For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.

Techniques: Activation Assay, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

doi: 10.1186/s12964-022-00879-3

Figure Lengend Snippet: The suppression of ezrin inhibited the LPS-induced production of cytokines. A549 cells were pre-treated with Y-27632 and subjected to LPS. The release of TNF-α, IL-1β, and HMGB1 into the supernatants was then measured by ELISA ( a – c ). The expression level of HMGB1 in cell lysates was measured by western blotting ( d ). Cell viability was examined by CCK-8 assay ( e ). A549 cells were transfected with ezrin-specific siRNA; TNF-α, and IL-1β expression was measured by ELISA ( f – h ), and the cellular levels of HMGB1 was measured by western blotting ( i ). Data are expressed as mean ± SD of triplicate samples. * p < 0.05 versus control group, # p < 0.05 versus LPS group

Article Snippet: For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, CCK-8 Assay, Transfection, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

doi: 10.1186/s12964-022-00879-3

Figure Lengend Snippet: A schematic summary of the ROCK-ezrin pathway in response to LPSs

Article Snippet: For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively.

Techniques: