Journal: Nucleic Acids Research

Article Title: Human snRNA genes use polyadenylation factors to promote efficient transcription termination

doi: 10.1093/nar/gkt892

Figure Lengend Snippet: Human snRNA genes do not use an Nrd1-like pathway to terminate transcription in vivo . ( A ) Western blot analysis of HeLa whole-cell extracts from control cells or cells transfected with an siRNA specific for SCAF8, senataxin and Xrn2. Antibodies used for detection were α-SCAF8 (left), α-Senataxin (middle) and α-Xrn2 (right). α-TATA-binding protein was used as a control for protein levels. ( B ) Schematic of the β-actin gene with the location of the primers used in ChIP assays indicated. The 5′ pause is described in . The arrow represents the TSS, exons are denoted by boxes and the position of the poly A site (pA) is indicated by a vertical line. Graphs represent results of pol II ChIP analysis before and after siRNA-mediated knockdown of Xrn2 (left graph), Senataxin (middle graph) and SCAF8 (right graph). ( C ) Schematics of the U1 and U2 snRNA genes are labelled as in . Graphs represent results of pol II ChIP analysis of the U1 (left panel) and U2 (right panel) snRNA genes before and after siRNA-mediated knockdown of Xrn2 (top), Senataxin (middle) and SCAF8 (bottom). Levels of pol II quantitated in (A) and (B), in control and knockdown cells, are normalized to levels quantitated on a non-transcribed region of the genome (120 bp region ∼2 kb upstream of the U2 snRNA gene). Error bars represent standard deviation of at least three independent experiments.

Article Snippet: Antibodies used in western blot studies were purchased from the following manufacturers: anti-SCAF8 (Bethyl, A301-036 A), anti-Senataxin (Bethyl, A301-105 A), anti-Int5 (Bethyl, A301–268 A), anti-Int9 (Bethyl, A300-412 A) and anti-Int11 (abcam, ab75276); anti-Xrn2 was a gift from Natalie Gromak ( ).

Techniques: In Vivo, Western Blot, Transfection, Binding Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Human snRNA genes use polyadenylation factors to promote efficient transcription termination

doi: 10.1093/nar/gkt892

Figure Lengend Snippet: Transcription termination pathways used by pol II transcribing human mRNA and snRNA genes. Termination factors that are common between both genes types are labelled in the same colour. Pol II CTD phosphorylation state is indicated as Phospho-Ser2 (S2P) and Phospho-Ser7 (S7P) ( A ). For protein-coding genes, the CPSF complex and the CstF complex recognize the poly A signal (AAUAAA) and U/GU-rich elements, respectively, as they emerge from the elongation complex. The CPSF-73 subunit, aided by CPSF-100, catalyzes cleavage at the poly A site (indicated by a lightning bolt) creating an entry site for the 5′–3′ exoribonuclease, Xrn2. Xrn2 degrades the downstream RNA product and displaces pol II. The CTD interacting domain of Pcf11 associates with the S2P form of the pol II CTD and bridges the polymerase with the nascent transcript, leading to pol II pausing and dismantling of the elongation complex. ( B ) The gene-specific PTF complex is recruited to the snRNA promoter and clears the transcription unit of nucleosomes to facilitate passage of pol II. The NELF complex is recruited at the end of the nucleosome-depleted region to terminate pol II where nucleosomes are encountered. CPSF-73 is not recruited to human snRNA genes but instead the Integrator complex directs cleavage upstream of the 3′ box processing element (indicated by a lightning bolt). The Int11 subunit is likely to catalyze the cleavage reaction with the help of Int9, as these subunits are homologous to CPSF-73 and CPSF-100, respectively. Thus, Integrator substitutes for the CPSF complex on snRNA genes. Ssu72 and Pcf11 are recruited to snRNA genes but do not have a major function in snRNA 3′-end processing. Instead, Ssu72 and Pcf11 act as transcription terminators for this gene class. CstF64 is also recruited, but its role in snRNA processing and transcription termination is not clear. Xrn2 is not implicated in snRNA transcription termination, but as cleavage upstream of the 3′ box would create an entry site for a 5′–3′ exoribonuclease, we cannot rule out that an alternative enzyme is recruited to promote termination.

Article Snippet: Antibodies used in western blot studies were purchased from the following manufacturers: anti-SCAF8 (Bethyl, A301-036 A), anti-Senataxin (Bethyl, A301-105 A), anti-Int5 (Bethyl, A301–268 A), anti-Int9 (Bethyl, A300-412 A) and anti-Int11 (abcam, ab75276); anti-Xrn2 was a gift from Natalie Gromak ( ).

Techniques:

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control

Journal: Oncology Letters

Article Title: Scutellarein suppresses Aβ-induced memory impairment via inhibition of the NF-κB pathway in vivo and in vitro

doi: 10.3892/ol.2019.10274

Figure Lengend Snippet: Scue attenuates Alzheimer's disease-like pathology in rats. (A) Latency of crossings and (B) average number of crossings in the Morris water maze spatial probe test of memory. Hippocampal expression of (C) Aβ, (D) SOD, (E) MDA and (F) Ach. (G) Nissl staining in the hippocampus. Scale bar, 50 µm. The expression levels of (H) Bcl-2, Bax and cleaved-caspase-3 were detected by western blotting. Densitometry analyses were conducted using β-actin as an internal reference. Representative results from repeated experiments are presented and data are presented as the mean ± standard deviation. n=6. **P<0.01, ***P<0.001 vs. the control group; # P<0.05, ## P<0.01, ### P<0.001 vs. the Aβ group. ^ P<0.05, ^^ P<0.01 vs. the Scu group. Aβ, amyloid β; Scu, scutellarin; Scue, scutellarein; SOD, superoxide dismutase; MDA, malondialdehyde; Ach, acetylcholine; Bcl-2, B cell lymphoma-2; Bax, apoptosis regulator BAX.

Article Snippet: The membranes were incubated with primary antibodies against nuclear factor of κ-light polypeptide gene enhancer in B cells inhibitor α [IκBα; cat. no. YS-(kt)-0907, phospho (p)-IκBα (cat. no. YS-KT1551) (Shanghai Yansheng Biochemical Reagent Co., Ltd., Shanghai, China), NF-κB, cleaved caspase-3 (cat. no. bs-0081R), B cell lymphoma-2 (Bcl-2; cat. no. bs-20351R), apoptosis regulator BAX (Bax; cat. no. bs-4564R), β-actin (cat. no. bs-0061R) and lamin-A (cat. no. bs-1839R) (1:1,000; BIOSS) overnight at 4°C.

Techniques: Expressing, Staining, Western Blot, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Girdin regulates the migration and invasion of glioma cells via the PI3K-Akt signaling pathway

doi: 10.3892/mmr.2015.4049

Figure Lengend Snippet: Girdin silencing inhibits the expression and activity of MMP-2 and MMP-9. (A and B) Changes in the mRNA levels of MMP-2 and MMP-9 were measured using reverse transcription-quantitative polymerase chain reaction following transfection. The relative mRNA expression levels were calculated using the 2 −ΔΔCt method. (C and D) Following transfection, changes in the protein levels of MMP-2 and MMP-9 were detected using western blot analysis. (E and F) Following transfection, gelatin zymography was performed to detect changes in the activities of MMP-2 and MMP-9. Each experiment was repeated three times. The experimental results are presented as the mean ± standard deviation. ** P<0.01, compared with the NC group. shRNA, short hairpin RNA; NC, negative control.

Article Snippet: Following washing with Tris-buffered saline with 0.05% Tween-20 (TBST), the membranes were incubated with the following primary antibodies at 4°C overnight: Rabbit anti-human polyclonal antibody against girdin (1:500 diluted; cat. no. bs-5150R; Bioss, Beijing, China); rabbit anti-human polyclonal antibody against MMP-2 (1:1,000 diluted; cat. no. WL0657); rabbit anti-human polyclonal antibody against MMP-9 (1:1,000 diluted; cat. no. WL0884); rabbit anti-human polyclonal antibody against P85α (1:1,000 diluted; cat. no. WL0191); rabbit anti-human polyclonal antibody against P110α (1:1,000 diluted; cat. no. WL0339); rabbit anti-human polyclonal antibody against AKT (1:1,000 diluted; cat. no. WL0003); rabbit anti-human polyclonal antibody against p-AKT (1:1,000 diluted; cat. no. WLP001).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Zymography, Standard Deviation, shRNA, Negative Control

Journal: Canadian Respiratory Journal

Article Title: Role of Erythromycin-Regulated Histone Deacetylase-2 in Benign Tracheal Stenosis

doi: 10.1155/2020/4213807

Figure Lengend Snippet: The expression of HDAC2 in the different groups determined by immunofluorescence. Image scale bar, 100 µ m. Red fluorescence (Cy3 staining) for the detection of the target protein HDAC2, blue fluorescence (DAPI staining) for the nucleus. Data are presented as the mean ± standard deviation in the corresponding histogram. ∗ P < 0.05 vs. the control group. Control: rabbit tracheal stenosis model without treatment; NS: rabbit tracheal stenosis model treated with penicillin; ERY: rabbit tracheal stenosis model treated with erythromycin; Budesonide: rabbit tracheal stenosis model treated with budesonide; Vorinostat: rabbit tracheal stenosis model treated with vorinostat. HDAC2, histone deacetylase-2; IF, immunofluorescence.

Article Snippet: Erythromycin enteric-coated tablets (H42021990, Yichang Humanwell Pharmaceutical Co., LTD.); Vorinostat Capsules (180509, Beijing Hengrui Kangda Medical Science and Technology Development Co., Ltd.); Budesonide (AstraZeneca 8339000); Rabbit Anti-Collagen III Polyclonal Antibody (bs-10423R, Bioss); Rabbit Anti-Collagen I Polyclonal Antibody (bs-0549R, Bioss); Rabbit Anti-HDAC2 Polyclonal Antibody (bs-1813R, Bioss); Rabbit VEGF ELISA kit (MM-021001); Rabbit TGF- β 1 ELISA kit (MM-3684001); Rabbit Polyclonal Anti-VEGF (bs-1313R, Bioss, 1/500–1/2000); Rabbit Polyclonal Anti-TGF β 1 (bs-0086R, Bioss, 1/500–1/2000); Rabbit monoclonal Anti-IL-8 (ab34100, abcam, 1/1000); Rabbit Polyclonal Anti-HDAC2 (OmnimAbs, OM105905, 1/500–1/2000); fluorescence microscope (CKX53, OLYMPUS); Microplate Reader (RT-6100, Rayto); Protein vertical electrophoresis instrument (DYY-6C, Beijing 61 instrument factory); Ultra High Sensitivity Chemiluminescence Imaging System (Chemi DocTM XRS+, Bio-Rad Shanhhai Laboratories).

Techniques: Expressing, Immunofluorescence, Fluorescence, Staining, Standard Deviation, Histone Deacetylase Assay

Journal: International Journal of Molecular Sciences

Article Title: Effects of Periostracum Cicadae on Cytokines and Apoptosis Regulatory Proteins in an IgA Nephropathy Rat Model

doi: 10.3390/ijms19061599

Figure Lengend Snippet: TLR4, MCP-1, IgA, and caspase-3 expression in rat kidney tissues. ( a ) TLR4, IgA, MCP-1, and caspase-3 expression levels were analyzed by Western blotting. ( b ) Quantitative analysis of TLR4, IgA, MCP-1, and caspase-3 protein levels. A: Healthy, normal controls; B: IgAN model group; C: prednisone acetate group; D: tripterygium glycoside tablet group; E–G: treatment group that received 0.5, 1, and 2 g/kg periostracum cicadae. Data represent the mean ± standard deviation (** p < 0.01 vs model group).

Article Snippet: Rabbit anti-TLR4 polyclonal antibody (Boster Biological Technology, Pleasanton, CA, USA), rabbit anti-IgA polyclonal antibody (Boster Biological Technology), rabbit anti-MCP-1 polyclonal antibody (Boster Biological Technology), and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (Boster Biological Technology) were used in the detection of the corresponding target proteins.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Biotechnology letters

Article Title: Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

doi: 10.1007/s10529-014-1555-9

Figure Lengend Snippet: VEGF (26 aM - 2.6 pM) was spiked into 50% BAL fluid (in PBS), and incubated with polyclonal anti-VEGF antibody functionalized magnetic particles. The biotinylated antibody/Neutravidin/phage affinity reagent was added, followed by washing. The sample was then analyzed using real-time PCR with phage-specific primers (n=6, error bars = ±1 SD, Non-template control Ct ≥34). The No-VEGF control (Ct =22.67±0.55) is shown as a solid line with +/− one standard deviation represented by dotted lines.

Article Snippet: The particles were washed, resuspended in coating buffer (1×10 7 particles in 1 ml 0.1 M sodium borate, pH 9.5) and mixed with 240 μl polyclonal anti-human Vascular Endothelial Growth Factor (VEGF) antibody (1 mg/ml, AB-293-NA, R&D Systems) and 240 μl 3 M (NH 4 ) 2 SO 4 .

Techniques: Incubation, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation

doi: 10.3389/fimmu.2020.576658

Figure Lengend Snippet: Emission of immunogenic cell death markers induced by combined adenoviral p14ARF + IFNβ gene transfer. SK-MEL-147 cells transduced as previously described, incubated for 48h h before cells and supernatants were collected for ICD assays. (A) Calreticulin exposure was assessed by flow cytometry after specific antibody staining. Representative dot plots and a bar graph showing the mean and standard deviation from three independent tests with three technical replicates each. (B) Supernatant from the same cultures were collected and evaluated for ATP secretion using a luciferase-based assay (RLU, relative light units). Data represent the mean and standard deviation from at least three independent experiments. (C) Detection of secreted IFNβ protein from cell supernatant by ELISA. Data represent the mean and standard deviation from at least three independent experiments. For both (A–C) , statistical analyses were performed using one-way ANOVA test followed by the Bonferroni post-test. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Article Snippet: For the evaluation of calreticulin exposure through flow cytometry, cells were probed with rabbit anti-calreticulin antibody (Novus Biologicals, Littleton, CO, USA), followed by the Alexa488-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific).

Techniques: Incubation, Flow Cytometry, Staining, Standard Deviation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: The Journal of International Medical Research

Article Title: Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury

doi: 10.1177/0300060520919592

Figure Lengend Snippet: Primer sequences for real-time quantitative polymerase chain reaction analyses.

Article Snippet: Mouse anti-rat MMP9 (cat. no. NBP2-13173SS) and TIMP2 antibodies (cat. no. NBP1-42375) were purchased from Novus Biologicals Inc. (Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: The Journal of International Medical Research

Article Title: Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury

doi: 10.1177/0300060520919592

Figure Lengend Snippet: Lung MMP and TIMP mRNA and protein expression. Changes in MMP2 (a), TIMP2 (b), MMP2/TIMP2 (c), MMP9 (d), TIMP1 (e), and MMP9/TIMP1 (f) in all groups treated with or without LPS. (g) Protein expression in each group as detected by western blotting. LPS 2h, 6h, 12h, and 24h indicate 2, 6, 12, and 24 hours after LPS injection in the acute lung injury group, respectively. Data are shown as mean ± standard deviation (n = 6 per group). * P < 0.05 vs. control, # P < 0.05 vs. LPS 2h. Control, normal control group; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; LPS, lipopolysaccharide

Article Snippet: Mouse anti-rat MMP9 (cat. no. NBP2-13173SS) and TIMP2 antibodies (cat. no. NBP1-42375) were purchased from Novus Biologicals Inc. (Littleton, CO, USA).

Techniques: Expressing, Western Blot, Injection, Standard Deviation, Control

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: BMC Infectious Diseases

Article Title: Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

doi: 10.1186/1471-2334-9-186

Figure Lengend Snippet: ELISA for total IgG antibody levels from acute (AC), indeterminate (IND) and cardiac chronic (CCC) chagasic patients and healthy individuals (HD) against T. cruzi lysate (2A), KMP-11 (2B), truncated T. cruzi HSP-70 (TcHSP-70T) (2C), and T. rangeli HSP-70 (2D) recombinant proteins . Values are given as optical densities at 405 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent mean and standard deviation values, the mean being the larger one. Statistically significant differences among chagasic groups are represented by an asterisk.

Article Snippet: Samples were diluted at 1:50 for all assays and anti-human IgG1, IgG2, IgG3 and IgG4 peroxidase conjugate (MP Biomedicals, Inc. Ohio, USA) were diluted at 1:500.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: BMC Infectious Diseases

Article Title: Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

doi: 10.1186/1471-2334-9-186

Figure Lengend Snippet: IgG isotype profile against T. cruzi lysate . IgG1 (4A), IgG2 (4B), IgG3 (4C) and IgG4 (4D) isotype levels for patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent mean and standard deviation values, the mean being the larger one.

Article Snippet: Samples were diluted at 1:50 for all assays and anti-human IgG1, IgG2, IgG3 and IgG4 peroxidase conjugate (MP Biomedicals, Inc. Ohio, USA) were diluted at 1:500.

Techniques: Standard Deviation

Journal: BMC Infectious Diseases

Article Title: Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

doi: 10.1186/1471-2334-9-186

Figure Lengend Snippet: IgG isotypes profile against T. cruzi KMP-11 recombinant protein . IgG1 (5A), IgG2 (5B), IgG3 (5C) and IgG4 (5D) isotype levels for patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent the mean and standard deviation values, the mean being the larger one.

Article Snippet: Samples were diluted at 1:50 for all assays and anti-human IgG1, IgG2, IgG3 and IgG4 peroxidase conjugate (MP Biomedicals, Inc. Ohio, USA) were diluted at 1:500.

Techniques: Recombinant, Standard Deviation

Journal: BMC Infectious Diseases

Article Title: Characterising the KMP-11 and HSP-70 recombinant antigens' humoral immune response profile in chagasic patients

doi: 10.1186/1471-2334-9-186

Figure Lengend Snippet: IgG isotypes profile against T. rangeli HSP-70 recombinant protein . IgG1 (6A), IgG2 (6B), IgG3 (6C) and IgG4 (6D) isotype levels of patients in acute (AC), indeterminate (IND) and cardiac chronic phase (CCC) and healthy individuals (HD). Values are given as optical densities at 492 nm. The dotted line represents the cut-off values based on the mean of healthy individual values plus 3 standard deviations. Horizontal lines on each group represent the mean and standard deviation values, the mean being the larger one.

Article Snippet: Samples were diluted at 1:50 for all assays and anti-human IgG1, IgG2, IgG3 and IgG4 peroxidase conjugate (MP Biomedicals, Inc. Ohio, USA) were diluted at 1:500.

Techniques: Recombinant, Standard Deviation