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: Acta biochimica et biophysica Sinica

Article Title: The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression.

doi: 10.1093/abbs/gmr094

Figure Lengend Snippet: Figure 2 The expression and distribution of ezrin and merlin in HUVECs The cells were fixed and visualized by fluorescence microscopy (400). Immunofluorescence stains was shown with red channel. (A) The expression and distribution of ezrin in HUVECs. (B) The expression and distribution of merlin in HUVECs.

Article Snippet: EBM-2 and EGM-2 media were purchased from Lonza (Cambrex, USA); Brdu kits were purchased from Chemicon (Billerica, USA); rabbit anti-human ezrin antibody, rabbit anti-phospho ezrin (Thr567) antibody, rabbit anti-human merlin antibody, and rabbit anti-phospho merlin (Ser518) were obtained from Cell Signaling Technology (Beverly, USA). siRNAs were designed and synthesized by Ambion (Austin, USA).

Techniques: Expressing, Fluorescence, Microscopy, Immunofluorescence

Journal: Acta biochimica et biophysica Sinica

Article Title: The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression.

doi: 10.1093/abbs/gmr094

Figure Lengend Snippet: Figure 3 The effects of oHA and nHA on ezrin/merlin mRNA expression in HUVECs After treatment with 10 mg/ml oHA or 200 mg/ml nHA for different time points, total RNA was extracted and ezrin/merlin mRNA expression was detected by RT–PCR. GAPDH was used a reference. (A, B) The expression of ezrin mRNA after induction by oHA and nHA in HUVECs. (C, D) The expression of merlin mRNA after induction by oHA and nHA in HUVEC. Representative image of three independent experiments was shown. *P , 0.05 vs. PBS group.

Article Snippet: EBM-2 and EGM-2 media were purchased from Lonza (Cambrex, USA); Brdu kits were purchased from Chemicon (Billerica, USA); rabbit anti-human ezrin antibody, rabbit anti-phospho ezrin (Thr567) antibody, rabbit anti-human merlin antibody, and rabbit anti-phospho merlin (Ser518) were obtained from Cell Signaling Technology (Beverly, USA). siRNAs were designed and synthesized by Ambion (Austin, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Acta biochimica et biophysica Sinica

Article Title: The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression.

doi: 10.1093/abbs/gmr094

Figure Lengend Snippet: Figure 4 The effects of oHA and nHA on ezrin/merlin protein expression and their phosphorylation in HUVECs HUVECs were treated with 10 mg/ml oHA or 200 mg/ml nHA for different time points, and total cell lysates were extracted. Protein expression and phosphorylation of ezrin and merlin were examined by western blot using specific antibodies. (A, B, C) The expression of ezrin protein and p-ezrin (Thr567) of HUVECs stimulated by oHA and nHA for different time points. (D, E, F) The expression of merlin protein and p-merlin (Ser518) of HUVECs after induction by oHA and nHA for different time points. Representative image of three independent experiments was shown. GAPDH was used as a reference. *P , 0.05 vs. PBS group.

Article Snippet: EBM-2 and EGM-2 media were purchased from Lonza (Cambrex, USA); Brdu kits were purchased from Chemicon (Billerica, USA); rabbit anti-human ezrin antibody, rabbit anti-phospho ezrin (Thr567) antibody, rabbit anti-human merlin antibody, and rabbit anti-phospho merlin (Ser518) were obtained from Cell Signaling Technology (Beverly, USA). siRNAs were designed and synthesized by Ambion (Austin, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression.

doi: 10.1093/abbs/gmr094

Figure Lengend Snippet: Figure 5 Effects of oHA and nHA on HUVECs proliferation after silencing of ezrin/merlin After HUVECs were transfected with siControl, three siRNA-ezrin or siRNA-merlin sequences for different time points, protein expression of ezrin (A) and merlin (B) was detected by western blot analysis. GAPDH was used as a reference. After silencing of ezrin (C) or merlin (D), HUVECs were stimulated with PBS, 10 mg/ml oHA and 200 mg/ml nHA for 72 h. The number of viable cells was determined using BrDu ELISA method. PBS and siControl are used as controls, respectively. Data were expressed as mean+SD from three different experiments.

Article Snippet: EBM-2 and EGM-2 media were purchased from Lonza (Cambrex, USA); Brdu kits were purchased from Chemicon (Billerica, USA); rabbit anti-human ezrin antibody, rabbit anti-phospho ezrin (Thr567) antibody, rabbit anti-human merlin antibody, and rabbit anti-phospho merlin (Ser518) were obtained from Cell Signaling Technology (Beverly, USA). siRNAs were designed and synthesized by Ambion (Austin, USA).

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

Journal: Acta biochimica et biophysica Sinica

Article Title: The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression.

doi: 10.1093/abbs/gmr094

Figure Lengend Snippet: Figure 6 The effects of oHA and nHA on ezrin/merlin mRNA expression after silencing of ezrin/merlin After ezrin- or merlin-targeting siRNA was transfected into HUVECs, 10 mg/ml oHA or 200 mg/ml nHA was added. After incubation with oHA/nHA for different time points, total RNA was extracted and ezrin/merlin mRNA expression was determined by RT–PCR. (A, B) The effects of oHA and nHA on merlin mRNA expression of HUVECs after transfection of ezrin-targeting siRNA. (C, D) The effects of oHA and nHA on ezrin mRNA expression of HUVECs after transfection of merlin-targeting siRNA. GADPH is used as a reference.

Article Snippet: EBM-2 and EGM-2 media were purchased from Lonza (Cambrex, USA); Brdu kits were purchased from Chemicon (Billerica, USA); rabbit anti-human ezrin antibody, rabbit anti-phospho ezrin (Thr567) antibody, rabbit anti-human merlin antibody, and rabbit anti-phospho merlin (Ser518) were obtained from Cell Signaling Technology (Beverly, USA). siRNAs were designed and synthesized by Ambion (Austin, USA).

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Acta biochimica et biophysica Sinica

Article Title: The influence of hyaluronic acid on vascular endothelial cell proliferation and the relationship with ezrin/merlin expression.

doi: 10.1093/abbs/gmr094

Figure Lengend Snippet: Figure 7 The effects of oHA and nHA on ezrin/merlin protein expression and their phosphorylation after silencing of ezrin/merlin After transfection with siRNA-ezrin or siRNA-merlin, HUVECs were treated with 10 mg/ml oHA or 200 mg/ml nHA for 72 h. Then, total cell lysates were extracted and ezrin/merlin protein expression and their phosphorylation levels were detected by western blot analysis. (A, B) The effects of oHA and nHA on merlin protein expression and phosphorylation of HUVECs after transfection with siRNA-ezrin before and 72 h after adding ezrin siRNA. (C, D) The effect of oHA and nHA on ezrin protein expression and phosphorylation of HUVECs after transfection with siRNA-ezrin. GADPH is used as a reference.

Article Snippet: EBM-2 and EGM-2 media were purchased from Lonza (Cambrex, USA); Brdu kits were purchased from Chemicon (Billerica, USA); rabbit anti-human ezrin antibody, rabbit anti-phospho ezrin (Thr567) antibody, rabbit anti-human merlin antibody, and rabbit anti-phospho merlin (Ser518) were obtained from Cell Signaling Technology (Beverly, USA). siRNAs were designed and synthesized by Ambion (Austin, USA).

Techniques: Expressing, Phospho-proteomics, Transfection, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Lyn prevents aberrant inflammatory responses to Pseudomonas infection in mammalian systems by repressing a SHIP-1-associated signaling cluster

doi: 10.1038/sigtrans.2016.32

Figure Lengend Snippet: Lyn regulates the IL-6/STAT3 signaling pathway by binding to IL-6R and Ezrin. ( a ) MH-S cells were transfected with control siRNA and Lyn siRNA at 5 pM for 48 h, respectively. The cells were then infected with PAO1 at an MOI of 20:1 for 30 min. IL-6 levels in supernatant were detected by enzyme-linked immunosorbent assay. ( b ) Expression of IL-6 detected by quantitative reverse transcription-PCR. ( c ) Expression of IL-6 and IL-6R was detected by immunoblotting. ( d ) Expression and phosphorylation of STAT3 were measured by immunoblotting. ( e ) Association of Lyn, IL-6R and Ezrin in Lyn-silenced and control MH-S cells was determined by immunoblotting with the indicated Abs for immunoprecipitation and detection in control cells and 30 min post PAO1-infected cells. ( f ) GST-tagged Lyn peptide fragments were used to study in vitro association of Lyn with IL-6R. ( g ) MH-S cells infected with PAO1 at an MOI of 20:1 for 30 min and lysed for pull-down assay. GST-Lyn 1–230 (SH2 and SH3). ( h , i ) MH-S cells were transfected with control siRNA and Ezrin siRNA at 5 pM for 48 h, respectively ( h ). Ezrin-silenced and control MH-S cells infected with PAO1 at an MOI of 20:1 for 30 min. Expression and phosphorylation of STAT3 measured by immunoblotting ( i ). ( j ) Cell viability of the indicated MH-S cells determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay. Data (mean±s.e.m.) are representative of three independent experiments (one-way analysis of variance with Tukey’s post hoc ; * P ⩽0.05; ** P <0.01).

Article Snippet: Mouse monoclonal Abs against GAPDH (SC-47724), STAT3 (SC-8019), p-STAT3 (SC-8059), Ezrin (SC-58758), IL-6R (SC-374259), GST (SC-374171), SHIP-1 (SC-271426), NF-κB p50 (SC-166588), p-NF-κB p50 (SC-271908), p-p38 (SC-166182), p-ERK (SC-7383), TLR4 (SC-293072) and p-Akt (SC-293125), polyclonal Abs against rabbit polyclonal Abs against Lyn (SC-28790), and goat polyclonal Abs against IL-6 (SC-1265) were obtained from Santa Cruz Biotechnology.

Techniques: Binding Assay, Transfection, Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription, Western Blot, Phospho-proteomics, Immunoprecipitation, In Vitro, Pull Down Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Lyn prevents aberrant inflammatory responses to Pseudomonas infection in mammalian systems by repressing a SHIP-1-associated signaling cluster

doi: 10.1038/sigtrans.2016.32

Figure Lengend Snippet: PAO1-induced MAPK phosphorylation is downregulated in SHIP-1 deficiency AMs. ( a ) MH-S cells were also transfected with Lyn siRNA alone or together with SHIP-1 siRNA at 5 pM for 48 h. Lyn-silenced, Lyn and SHIP-1 double-silenced, and control MH-S cells were then infected with PAO1 at an MOI of 20:1 for 0, 15, 30 and 60 min. Phosphorylation of p38 and ERK1/2 was measured by immunoblotting. ( b , c ) Expression of TLR4 ( b ) and phosphorylation of Akt ( c ) were measured by immunoblotting in MH-S cells after PAO1 infection (MOI=20:1, 30 min). ( d ) These cells were treated with Akt inhibitor (20 μ m ) before and during PAO1 infection (MOI=20:1, 30 min). Phosphorylation of Akt, p38 and ERK1/2 was measured by immunoblotting. C, control; I, inhibitor. Data are representative of three independent experiments. ( e ) Schematic representation of Lyn’s role in P. aeruginosa infection. Upon PAO1 infection, free Lyn binds IL-6R and Ezrin, to inhibit STAT3 activation and further impairs SHIP-1 activation. Inhibition of SHIP-1 dampens NF-κB activation and its downstream pro-inflammatory cytokines production, as well as reduction MAPK activation by inducing Akt phosphorylation.

Article Snippet: Mouse monoclonal Abs against GAPDH (SC-47724), STAT3 (SC-8019), p-STAT3 (SC-8059), Ezrin (SC-58758), IL-6R (SC-374259), GST (SC-374171), SHIP-1 (SC-271426), NF-κB p50 (SC-166588), p-NF-κB p50 (SC-271908), p-p38 (SC-166182), p-ERK (SC-7383), TLR4 (SC-293072) and p-Akt (SC-293125), polyclonal Abs against rabbit polyclonal Abs against Lyn (SC-28790), and goat polyclonal Abs against IL-6 (SC-1265) were obtained from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Transfection, Control, Infection, Western Blot, Expressing, Activation Assay, Inhibition

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