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: International journal of oncology

Article Title: Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

doi: 10.3892/ijo.2014.2397

Figure Lengend Snippet: Figure 3. Reduced production of VEGF and transcription of both VEGF-A and VEGF-B by C. militaris extract. (A) Dot blot-human angiogenesis array of MeWo culture medium after C. militaris extract or cordycepin treatment. Culture media of MeWo cells incubated with control, 5% C. militaris extract, 20 µM cordycepin, or 100 µM cordycepin for 48 h were used to detect the production of various angiogenesis-related proteins. All treatment groups showed lower production of angiogenin, TIMP-1, PDGF-AA, and VEGF compared to the control group. Production of TIMP-1 and VEGF was especially reduced in the 5% C. militaris treatment group. (B and C) qPCR of VEGF-A and VEGF-B. Transcription levels of VEGF-A and VEGF-B were quantified from MeWo cells after incubation with either 5% C. militaris extract (B) or 20 µM cordycepin (C) for 48 h. Transcription levels of VEGF-A and VEGF-B were significantly decreased after treatment with 5% C. militaris extract, while only VEGF-A was decreased after treatment with 20 µM cordycepin. *P<0.05 significantly different from the control group. ns, not significant.

Article Snippet: Screening for angiogenesis-related factors produced by MeWo cells was carried out by using the Human angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA).

Techniques: Dot Blot, Incubation, Control

Journal: International journal of oncology

Article Title: Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells.

doi: 10.3892/ijo.2014.2397

Figure Lengend Snippet: Figure 6. Treatment with C. militaris extract reduced angiogenesis. (A) Decrease of FITC-lectin intensity after treatment with 5% C. militaris extract. Using the Cultrex Directed In Vivo Angiogenesis Assay (DIVAA) (Trevigen, Gaithersburg, MD, USA), angioreactors containing a mixture of BME and DDW or BME and 5% C. militaris extract were implanted subcutaneously into the flanks of mice for 16 days. After extraction, FITC-lectin intensity was measured using excitation 485 nm, emission 510 nm. (B) Chorioallantoic membrane (CAM) assay showed that neovascularization was inhibited by C. militaris extract. Eight days post-fertilized chicken egg shells were cut to place silicon rings on the CAM. The inner spaces of the silicon rings were filled with DDW or 50 µl of C. militaris extract. Egg shells were covered using transparent tape, and eggs were incubated for 48 h. Results were observed by decrease or increase of vascular branch formation from main capillary vessels.

Article Snippet: Screening for angiogenesis-related factors produced by MeWo cells was carried out by using the Human angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vivo, Angiogenesis Assay, Extraction, Membrane, Chick Chorioallantoic Membrane Assay, Incubation

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: HT-29 cells and HCPECs were stimulated with LPS (200 ng/ml) at various time points as indicated. ( A ) Representative western blot analysis of HT-29 cells. Phosphorylation and/or total protein levels of ERK3, ERK4, MK5 and JNK were monitored. Tubulin immunoblots and Ponceau S staining were employed to monitor equal loading. ( B ) Changes in the expression and phosphorylation of ERK3 protein are shown as a fold change after normalization with internal loading control. Each time point was normalized to unstimulated cells (0). Fold change values from three independent experiments (n = 3) are represented as mean ± SEM. ( C ) Activation kinetics of MK5 in HT-29 cells stimulated with LPS. Fold change in MK5 phosphorylation levels upon LPS stimulation normalized to total protein levels and expression levels of MK5 normalized to internal loading control are shown. Fold change values from three independent experiments (n = 3) are represented as mean ± SEM. ( D ) Quantitative RT-PCR analysis of ERK3 expression. Each biological replicate was measured in triplicates. Log2 fold change in gene expression is presented as mean ± SEM of three independent experiments (n = 3); *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Turkey’s post-test. ( E ) ERK3 protein stability was assessed by CHX chase at 0 hr, 0.5 hr, 1 hr, 2 hr, 3/4 hr and 6 hr in the presence and absence of LPS (30 min pre-treatment). Western blot analyses were performed and representative results are presented. ERK3 protein levels in respect to the untreated cells (-LPS, 0 hr) were calculated using ImageJ and data are presented as mean fold change ± SEM from three independent experiments (n = 3). ( F ) Graph presents ERK3 protein levels quantified in respect to the untreated cells (0) of unstimulated (-LPS) and LPS stimulated (+LPS) cells, respectively and data are presented as mean fold change ± SEM from three independent experiments (n = 3). ( G ) HCPECs were stimulated with LPS and immunoblot analyses of the phosphorylation and/or total protein levels of ERK3, MK5 and p38 were performed. Actin and Ponceau S staining were used as loading controls. ( H ) Plotted here are fold changes in expression of ERK3 protein. Results are shown as mean ± SEM fold change after normalization with the levels of internal loading control. Each time point was normalized in respect to unstimulated HCPECs (0). Data are a representative of three independent experiments (n = 3); *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Turkey’s post-test. ( I-K ) Plotted here are fold changes in the phosphorylation of ( I ) MK5 at T182, ( J ) ERK1/2 and ( K ) p38 in response to LPS stimulation normalized to the respective total protein levels as well as the expression levels of total proteins normalized in respect to the internal loading control. Each time point was normalized in respect to the unstimulated cells (0). Fold change values are presented as mean ± SEM from three independent experiments (n = 3). ( L ) Quantitative RT-PCR analysis of ERK3 mRNA expression levels. Log2 fold change in gene expression is presented as mean ± SEM of three independent experiments (n = 3); *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Turkey’s post-test. ( M-N ) LPS-mediated ubiquitination of endogenous ERK3 in ( M ) HT-29 cells and ( N ) HCPECs. HT-29 cells and HCPECs were seeded and treated as mentioned in the Materials and methods. Total cell lysates (TCL) and endogenous ERK3 immunoprecipitates (IP) were analyzed by immunoblotting. Levels of ERK3 and polyubiqutination were monitored. Actin and Ponceau S staining were used as loading controls for TCL western blot analysis. Results are representatives of at least two experiments showing the same tendency. ERK3 kinetics in response to other immune stimuli are presented in ). Figure 2—source data 1. Full membrane scans for western blot images for .

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Phospho-proteomics, Staining, Expressing, Control, Activation Assay, Quantitative RT-PCR, Gene Expression, Ubiquitin Proteomics, Membrane

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: ( A–B ) Western blot analysis of CaCo2 cells stimulated with LPS. CaCo2 cells were stimulated at indicated time points with LPS and activation status of ERK3 as well as the phosphorylation of p38 were analyzed by immunoblotting. Actin and Ponceau S staining were used as loading controls. ( A ) Representative western blot. ( B ) Changes in the expression and phosphorylation of ERK3 protein are shown as a fold change after normalization with internal loading control. Each time point was normalized to unstimulated cells. Fold change values from two (n = 2) independent experiments are represented and means were connected for better visualization. Figure 2—figure supplement 1—source data 1. Full membrane scans for western blot images for .

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Staining, Expressing, Control, Membrane

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: ( A-F ) HCPECs and HT-29 cells were stimulated for 0, 0.5, 1 and 2 hr with IL-1β, Pam3CSK4 and R848, cells were lysed for western blot analysis. Representative immunoblots depicting phosphorylation and total protein levels of ERK3 (S189)/ERK3 as well as the phosphorylation of p38 MAPK. Ponceau S staining and actin were used as loading controls.

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Phospho-proteomics, Staining

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet: ( A–D ) HCPECs were transfected with siRNA targeting ERK3 (siERK3) or control (siCo), 24 hr post-transfection, medium was exchanged for MEM without supplements and cells were stimulated with IL-1β or Pam3CSK4 for 24 hr. Supernatants were collected for IL-8 ELISA analysis and cells were subjected to immunoblotting. Levels of IL-8 are presented as mean concentration in pg/ml ± SEM from two biological replicates (n = 2). Knockdown efficiency was determined using ERK3 antibody and phosphorylation status of p38 and ERK1/2 was monitored. Actin and Ponceau S staining were used as loading controls. ( E-H ) Control (shCo) and ERK3-depleted (shERK3) HT-29 cells were seeded in 12-well plate. After cells reached 70–80% confluency, medium was exchanged for McCoy’s without serum and cells were stimulated with IL-1β or Pam3CSK4 for 24 hr. Supernatants were collected for IL-8 ELISA analysis and cells were subjected to western blot analysis. IL-8 concentration in pg/ml is presented as mean ± SEM of two biological replicates (n = 2). Expression levels of ERK3 were verified to determine knockdown efficiency and phosphorylation of p38 and ERK1/2 was monitored. Actin and Ponceau S staining are provided as loading controls. Figure 4—figure supplement 3—source data 1. Full membrane scans for western blot images for .

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Concentration Assay, Knockdown, Phospho-proteomics, Staining, Expressing, Membrane

Journal: eLife

Article Title: ERK3/MAPK6 controls IL-8 production and chemotaxis

doi: 10.7554/eLife.52511

Figure Lengend Snippet:

Article Snippet: Anti-ERK3 antibody (Cat# 4067), anti-MK5/MAPKAPK5 (D70A10) antibody (Cat# 7419), anti-V5-tag antibody (Cat# 13202), anti-p44/42 MAPK (ERK1/2) antibody (Cat# 9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cat# 9101L), anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (Cat# 9215), anti-p38 MAPK antibody (Cat# 9212), anti-IκBα (44D4) antibody (Cat# 4812), anti-phospho-SAPK/JNK (183/Y185) antibody (Cat# 9251), Normal Rabbit IgG antibody (Cat# 2729), anti-c-Jun (60A8) antibody (Cat# 9165) and Histone H3 (D1H2) antibody (Cat# 4499) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Concentration Assay, Recombinant, Sequencing, shRNA, CRISPR, Mutagenesis, Enzyme-linked Immunosorbent Assay, Cell Fractionation, Ab Array, Extraction, Activation Assay, Luciferase, Chemotaxis Assay, Neutralization, Control, Software, Staining, Fluorescence

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Definition of the earliest PanINs and their physical location in the pancreas of KC mice. A and F) The representative 3D imaging projection of the whole pancreas of 2 (A)‐ and 4 (F)‐week‐old Pdx1 ‐Cre; LSL‐ Kras G12D/+ (KC) transgenic mouse using 3D histological analysis. Red arrow: the early PanIN. Blue signal: the nucleus staining; White signal: CK19‐staining; Red signal: the blood vessel staining. Scale bar: 1 mm. B and G) Quantification of PanIN number in 2 (B)‐ and 4 (G)‐week‐old transgenic mice. Each dot represents the datum of one mouse. Values were presented as mean ± SD. C and H) The distribution of the earliest PanIN lesions from 45 and 24 of 2 (C)‐ and 4 (H) ‐week‐old KC mice, respectively. One yellow dot indicates one lesion. n = 109 (C); n = 211 (H). D and I) Stacked bar plot showing the percentage of PanIN in the pancreas's head, body, and tail of 2 (D)‐ and 4 (I)‐week‐old KC mice. E and J) Quantification of PanIN and islet association in the pancreas of 2 (E)‐ and 4 (J) ‐week‐old KC mice. An association is defined by the distance between lesion and islet within 300 µm.

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Imaging, Transgenic Assay, Staining

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Genetic alterations in the earliest PanIN and Muc4 up‐regulation cooperate with oncogenic Kras G12D for PanIN initiation. A) The most common genetic alterations in the earliest PanINs from the pancreas of 2‐week‐old KC mice. The data analyzed in this study were obtained from 21 lesions in 13 mice, which were compared to the normal pancreatic region within the same KC mouse. In addition, two control mice were included for comparison. To ensure accuracy, all mouse‐specific single nucleotide polymorphisms (SNPs) were excluded by referencing the UCSC Genome Browser website. B) The RNA‐seq data of the Muc4 gene and Sirpb1a , comparing the PanIN sample with the control sample from a 2‐week‐old KC mouse (M1376). C) The IHC staining of Ki67 proliferation marker. Representative images (left panel) and quantification (right panel) of Ki67 staining in 4‐week‐old control and KC mice. Bar, 50 µm. Each dot represents the datum of one mouse. Values were presented as mean ± SD, n = 11 mice. *, P <0.05; **, P <0.01; ***, P < 0.001 (two‐tailed Student's t‐test). D) Plots of the survival probability of pancreatic cancer patients with MUC4 (left panel) and SIRPB1 (right panel) mRNA expression levels from the Human Protein Atlas as shown in methods. P values are calculated by log‐rank test. E–H) The Alcian Blue staining and IHC analysis with anti‐Muc4 antibody in 4‐week‐old control and KC mice. Representative images of IHC staining with Alcain Blue and Muc4 E), quantification of Alcain Blue F) and Muc4 G) in the early PanIN cells. Bar, 50 µm. The H score of Muc4 expression in 4‐week‐old control and KC mice H). Each dot represents the datum of one mouse. Values were presented as mean ± SD, n = 11 mice. ****, P < 0.0001 (two‐tailed Student's t‐test). I and J) EpCAM + /Muc4 − ‐ or double EpCAM/Muc4‐positive pancreatic cells isolated by FACS from 4‐week‐old KC mice for spheroid formation analysis I) and soft agar colony formation analysis J). N indicates independent experiments, and n indicates total measurements in all experiments. Each dot represents the datum from one measurement. Values show mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). K and L) Primary acinar cells were isolated from 4‐week‐old control or KC mice for spheroid formation analysis K) and soft agar colony formation analysis L). N indicates independent experiments, and n indicates total measurements. Each dot represents the datum from one measurement. Values show mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Control, Comparison, RNA Sequencing, Immunohistochemistry, Marker, Staining, Two Tailed Test, Expressing, Isolation

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: αSMA + fibroblasts associate with the earliest PanIN cells to promote Kras G12D/+ pancreatic cell transformation and stemness properties. A) Representative image of 3D histology‐detected the earliest PanINs in the whole pancreas of 2‐week‐old KC mice. Blue signal: the nucleus staining; White signal: CK19‐staining (PanIN cells); Green signal: αSMA staining (activated fibroblasts). Bar, 100 µm. B) Quantification of the percentage of the earliest PanINs associated with αSMA + fibroblasts. C) Representative images of the IHC staining of fibroblasts with anti‐αSMA antibody in 4‐week‐old KC mice (left panel) and quantification of the percentage of early PanINs associated with αSMA + fibroblasts (right panel). Bar, 50 µm. D) Close contact with fibroblasts promotes acinar‐to‐ductal metaplasia of Kras G12D/+ pancreatic acinar cells in 3D Matrigel coculture systems. X: Kras G12D/+ pancreatic acinar cells only (1000 cells). F: coculture with Kras +/+ fibroblasts (2000 cells). I: coculture with Kras +/+ islet cells (2000 cells). Upper panel: Representative images in coculture experiment in Kras G12D/+ pancreatic cells. Bar, 50 µm. Bottom panel: Quantification of cyst number. E and F) Close contact of Kras G12D/+ pancreatic acinar cells with fibroblasts promotes sphere formation. E) 1000 Kras G12D/+ pancreatic acinar cells labeled with CellTracker Green CMFDA were cocultured with 2000 Kras +/+ fibroblasts or 2000 Kras +/+ isle cells for 14 days in a 96‐well low attached plate. (Left panel) Representative images of the cocultured Kras G12D/+ pancreatic cells. (Right panel) Quantification of sphere number. Spheres with a diameter ≥ 100 µm were counted. F) 1000 Kras G12D/+ pancreatic acinar cells labeled with CellTracker Green CMFDA were cocultured with different ratios of Kras +/+ fibroblasts for 14 days in a 96‐well low attached plate. Spheres with a diameter ≥ 100 µm were counted. In the above experiments, N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). G) EpCAM + /PDGFRα + cell clusters derived from the pancreas of 4‐week‐old control or KC mice by MACS dissociation and FACS with EpCAM/PDGFRα antibodies for cyst formation analysis. Representative images of cysts without and with lumen were shown in the left panel. CK19 staining indicates pancreatic ductal cells, and αSMA staining indicates activated fibroblasts. Bar, 100 µm. The number of cysts was counted and compared in the right panel. N indicates independent experiment and n indicates cyst number. H) The same cell clusters as above, in addition two more controls including EpCAM + and non‐ EpCAM + /PDGFRα + clusters from KC mice, were used for sphere formation analysis. Representative images of spheres were shown in the upper panel and the number of spheres were counted (lower panel). Spheres with a diameter ≥ 100 µm were counted. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ***, P < 0.001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Transformation Assay, Staining, Immunohistochemistry, Labeling, Two Tailed Test, Derivative Assay, Control

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Muc4 overexpression in Kras G12D/+ pancreatic cells promotes fibroblast activation and recruitment in the earliest PanIN. A) The mRNA levels of Muc4, Muc1, and Muc5a in the EpCAM + /PDGFRα + clusters derived from the pancreas of 4‐week‐old KC mice were measured and compared with that of the control mice after normalized with the control gene, GAPDH . Primary mPSCs in all experiments were isolated from the pancreas of 4‐week‐old control mice. For chemotaxis analysis, mPSCs were labeled with CellTracker Green CMFDA for 10 mins before the experiment. B) Expression of ACTA2 gene in mPSC treated with conditional media derived from EpCAM + /Muc4 − or EpCAM + /Muc4 + pancreatic cells isolated by FACS from 4‐week‐old control or KC mice by qPCR. C) Left panel: Representative images of µslide chemotaxis analysis of mPSC after treated with conditional media as B). Right panel: Quantitation of the percentage of chemotaxis. D–F) EpCAM + /Muc4 − ‐ or EpCAM + /Muc4 + ‐pancreatic cells were cocultured with mPSC cells for cyst formation analysis D), sphere formation analysis E), and soft colony formation analysis F). For sphere formation analysis, sphere with a diameter ≥ 100 µm were counted. G) Expression of ACTA2 gene in mPSC treated with conditional media derived from pancreatic acinar cells isolated from 4‐week‐old control or KC mice by qPCR. These primary pancreatic acinar cells were infected with lentiviral GFP or lentiviral GFP‐MUC4 (MUC4/X). H) Left panel: Representative images of µslide chemotaxis analysis of mPSC after treated with conditional media as G). Right panel: Quantitation of the percentage of chemotaxis. I and J) Pancreatic acinar cells from KC or control mice were ectopically expressed GFP‐MUC4/X or GFP only and cocultured with mPSC cells for sphere formation analysis I) and soft colony formation analysis J). For soft agar colony formation analysis, the colony with a diameter ≥ 50 µm was counted. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. **, P < 0.01; ****, P < 0.0001 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Over Expression, Activation Assay, Derivative Assay, Control, Isolation, Chemotaxis Assay, Labeling, Expressing, Quantitation Assay, Infection, Two Tailed Test

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: Activin A from Muc4 overexpressed and Kras G12D/+ pancreatic cells facilitate fibroblast recruitment for PanIN formation. A) Cytokines analysis of conditional media from the cell clusters isolated from 4‐week‐old control or KC mice using RayBio® Mouse Biotin‐Label Based Antibody Array (Mouse L‐308 Array, Glass Slide). B) The effect of Activin A on mPSC chemotaxis was analyzed using μ‐Slide chemotaxis analysis. Representative images (left panel) and quantification (right panel) of the chemotaxis effect. C) Quantification of Activin A in the conditional media of EpCAM + /Muc4 − ‐ or EpCAM + /Muc4 + ‐pancreatic cells using ELISA analysis. D) Anti‐Activin A antibody abolishes mPSC chemotaxis. Conditional media from EpCAM + /Muc4 + ‐pancreatic cells were pre‐treated with or without 4 µg mL −1 of anti‐Activin A antibody for 30 mins and were subjected to µslide chemotaxis analysis. E) Quantification of Activin A in the conditional media of pancreatic acinar cells infected with lentiviral GFP or lentiviral GFP‐MUC4 (MUC4/X) using ELISA. F) Anti‐Activin A antibody abolishes mPSC chemotaxis. Conditional media from lentiviral GFP‐MUC4/X‐infected pancreatic acinar cells were pre‐treated with or without 4 µg mL −1 of anti‐Activin A antibody for 30 min and were subjected to μ‐Slide chemotaxis analysis. G) Increases of Activin A secretion of media from FACS‐isolated pancreatic cells cocultured with or without fibroblasts by ELISA analysis. H) Activin A secretion from Kras G12D/+ pancreatic acinar cells infected with the indicated lentivirus after cocultured with or without fibroblasts by ELISA analysis. In the above experiments, N indicates independent experiments, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two‐tailed Student's t‐test). I) Activin A mRNA expression in PanIN and PanIN‐associated fibroblasts in 4‐week‐old KC mice was detected by an Opal Multiplex IHC Assay. Representative IHC images (left panel) and quantification of Activin A expressed in early PanIN cells (right panel). Bar, 20 µm. White signal: DAPI; Cyan signal: Normal duct cells and PanIN cells stained with anti‐CK19 antibody; Red signal: αSMA + fibroblasts stained with anti‐αSMA antibody; Green dot signal: Activin A mRNA with dig‐labeled antisense mRNA probes. Values were presented as mean ± SD, PanIN lesion n = 29 from 12 KC mice. J) Sera from 4‐week‐old control or KC mice were used to perform Activin A ELISA analysis. N = 7 mice; two duplicate experiments for each mouse, n = total 14 repeated measurements. Each dot represents the datum from one measurement. Values were presented as mean ± SD. *, P < 0.05 (two‐tailed Student's t‐test).

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Isolation, Control, Ab Array, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Expressing, Multiplex Assay, Staining, Labeling

Journal: Advanced Science

Article Title: Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

doi: 10.1002/advs.202301240

Figure Lengend Snippet: The proposed model for the cooperation of oncogenic Kras G12D/+ , Muc4 overexpression, and fibroblast activation for PanIN initiation. A–E) 3‐week‐old KC mice were administered either Saline solution or a dose of 1 µg k −1 g follistatin (FST) for a duration of two weeks. After the two‐week treatment period, we performed integrated 3D/2D pancreas histology to assess fibroblast recruitment and PanIN formation. A) Representative images depicting 3D and 2D histology of the pancreas. In the 3D staining images, green color represents CK19 staining, indicating PanIN or ductal cells; red color represents αSMA staining, indicating activated fibroblasts; and white color represents DAPI staining, indicating nuclei. In the 2D IHC images, brown color represents αSMA staining, indicating activated fibroblasts. Scale bar: 100 µm. B) The percentage of lesions associated with fibroblast. Each dot represents the datum of one mouse. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. ***, P < 0.001 (two‐tailed Student's t‐test). C) The H score of αSMA + fibroblasts associated with lesion of specified mice. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. **, P < 0.01 (two‐tailed Student's t‐test). D) Representative lesion images (upper panel) and atrophy lesion quantification (lower panel) at KC mice treated with Saline solution or FST. Green color represents CK19 staining, indicating PanIN. Each dot represents the datum of one mouse. Values were presented as mean ± SD. Mouse number and lesion number are denoted by N and n, respectively. **, P < 0.01 (two‐tailed Student's t‐test). E) The percentage of lobe and lesions volume in KC mice treated with Saline solution or FST. Mouse number and lesion number are denoted by N and n, respectively. The total tissue volume calculated in the Saline solution group is 279.4 mm 3 , while in the FST group, it is 252.8 mm 3 . F) mPSCs were obtained from 4‐week‐old control mice and subjected to overnight infection with 10 MOI of the specified lentiviral shRNA. After one day of recovery from the virus infection, we selectively enriched lentiviral shRNA‐positive cells by applying puromycin selection at a concentration of 1 µg mL −1 for three days. Following a two‐day recovery period, the cells were subjected to Western blot analysis (upper panel) and cocultured with double EpCAM and Muc4‐positive pancreatic cells isolated from 4‐week‐old KC mice for the assessment of soft agar colony formation (lower panel). The number of colonies larger than 50 µm was quantified after a 14‐day coculture period. N indicates independent experiment, and n indicates total repeated measurements in all experiments. Each dot represents the datum from one measurement. Values were presented as mean ± SD. ****, P < 0.0001 (two‐tailed Student's t‐test). G) Proposed model of oncogenic Kras G12D ‐mediated PanIN initiation. The overexpression of Muc4 (Muc4/X) in Kras G12D/+ pancreatic cells enhances cell transformation and stimulates Activin A secretion, which in turn activates and recruits' fibroblasts. The activated fibroblasts further promotes Activin A secretion, elevates pancreatic cell transforming status, and enhances cancer stemness properties, thereby contributing to PanIN initiation. The inhibition of Activin A signaling using Follistatin (FST), an Activin A antagonist, effectively suppresses fibroblast recruitment and inhibits PanIN formation. Additionally, reducing Activin A expression in mPSCs through the use of lentiviral Inhba shRNA also impedes PanIN formation.

Article Snippet: After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ̊C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Techniques: Over Expression, Activation Assay, Saline, Staining, Two Tailed Test, Control, Infection, shRNA, Virus, Selection, Concentration Assay, Western Blot, Isolation, Transformation Assay, Inhibition, Expressing

Journal: Cell host & microbe

Article Title: Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation

doi: 10.1016/j.chom.2018.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-MEK 1⁄2 , Cell Signaling , Cat # 8727, RRID:AB_10829473.

Techniques: Virus, Isolation, Recombinant, Modification, Ab Array, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, BIA-KA, Extraction, Membrane, Protein Extraction, Luciferase, Endotoxin Assay, LDH Cytotoxicity Assay, shRNA, Control, Plasmid Preparation, Transgenic Assay, Knock-Out, Software

Journal: BMB Reports

Article Title: Regorafenib prevents the development of emphysema in a murine elastase model

doi: 10.5483/BMBRep.2023-0072

Figure Lengend Snippet: Effect of regorafenib on PPE-induced in inflammatory mediators in BAL fluid. (A) Representative membrane images of the antibody array. (B) Quantification of relative optical density for IL-1β, IL-6, CXCL1/KC, and TIMP-1 in the array membranes. (C-F) Measurement of IL-1β (C), IL-6 (D), CXCL1/KC (E), and TIMP-1 (F) levels in BAL fluid by ELISA. Error bars indicate the mean ± SEM. N = 8 per group, *P < 0.05, **P < 0.01, ****P < 0.0001, t -test.

Article Snippet: Anti-IL-6 antibody (ab208113) from abcam (Cambridge, UK), CXCL1 antibody (12335-1-AP) from Proteintech (Rosemont, IL, USA), and TIMP-1 antibody (#AF980) from R&D Systems were purchased respectively.

Techniques: Membrane, Ab Array, Enzyme-linked Immunosorbent Assay

Journal: BMB Reports

Article Title: Regorafenib prevents the development of emphysema in a murine elastase model

doi: 10.5483/BMBRep.2023-0072

Figure Lengend Snippet: Effect of regorafenib on PPE-induced expression of inflammatory mediators in lung parenchyme. (A) Experimental scheme. Mice were sensitized by treatment with regorafenib (5 mg/kg) for one day in the only PPE + Reg group. After PPE treatment, regorafenib was administered orally to mice for six days. On the seventh day, lung tissues were removed from mice, prepared in paraffin sections and stained with antibodies for inflammatory mediators. (B) Representative images of stained paraffin sections. Red arrows indicate the macrophages in inserted boxes. Scale bars indicate 50 μm. (C-E) Quantification of . IL-6 (C), CXCL1/KC (D), and TIMP-1 (E). Error bars indicate the mean ± SEM. N = 5 per group, *P < 0.05, **P < 0.01, t -test.

Article Snippet: Anti-IL-6 antibody (ab208113) from abcam (Cambridge, UK), CXCL1 antibody (12335-1-AP) from Proteintech (Rosemont, IL, USA), and TIMP-1 antibody (#AF980) from R&D Systems were purchased respectively.

Techniques: Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: Tumor-extrinsic discoidin domain receptor 1 promotes mammary tumor growth by regulating adipose stromal interleukin 6 production in mice

doi: 10.1074/jbc.RA117.000672

Figure Lengend Snippet: Identification of DDR1-dependent cytokines in SVF. a, representative images of the membrane-based antibody array using cell-free supernatant from WT- and KO-SVF. The boxed dots are a reference protein (blue) and IL-6 (red). b, mRNA levels of IL-6 in WT/KO-SVF by RT-qPCR. c, measurement of secreted IL-6 in cell-free supernatant from WT/KO-SVF by ELISA. d, mRNA levels of Cofilin in WT/KO-SVF by RT-qPCR. e, protein levels of DDR1 and Cofilin in three pairs of WT/KO SVF. GAPDH was a loading control. f, quantification of Cofilin protein level normalized by GAPDH. All mRNA analyses and ELISA were done with four pairs of WT and KO. Data represent mean ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The membrane was blocked using 5% BSA and immunoblotted with anti-DDR1 (D1G6) XP rabbit mAb (Cell Signaling Technology, catalog no. 5583), GAPDH ( 14 C10) rabbit mAb (Cell Signaling Technology, catalog no. 2118), Cofilin mouse mAb (Santa Cruz Biotechnology, catalog no. SC-53934), and α-tubulin mouse mAb (Calbiochem, catalog no. CP06).

Techniques: Membrane, Ab Array, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, 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: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 1. Induction of thymic stromal lymphopoietin (TSLP) mRNA and protein expression in mouse intervertebral disc cells via the NF-B pathway. A, Left, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml tumor necrosis factor (TNF), interleukin-1 (IL-1), and 1 g/ml lipopolysaccharide (LPS) for the indicated periods of time. RNA was then extracted, and real-time reverse transcription– polymerase chain reaction was performed using specific primers for TSLP and GAPDH. The ratio for expression of each gene to that of GAPDH was calculated, and the relative expression levels are shown. Right, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TNF, IL-1, IL-6, and transforming growth factor (TGF), and 1 g/ml LPS for 72 hours. The culture supernatants were collected, and TSLP concentrations were measured by enzyme-linked immunosorbent assay (ELISA). B, For immunohistochemical examination of mouse intervertebral disc tissue, tissue sections were stained with anti-mouse TSLP antibody as described in Materials and Methods. No positive immunohistologic cell staining following stimulation with TNF was seen in control IgG-treated sections (goat-Ab). C, Mouse intervertebral discs obtained from wild-type (WT), TNF receptor I (TNFRI)–deficient (RI-knockout [RIKO]), and TNFRII-deficient (RIIKO) mice were cultured in the presence or absence of 10 ng/ml TNF or 1 g/ml LPS for 72 hours. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. D, Mouse intervertebral discs obtained from wild-type mice were cultured in the presence or absence of 10 ng/ml TNF, IL-1, or 1 g/ml LPS with or without 1 M IMD-0354 or 10 M MG132 for 72 hours. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. Values in A, C, and D are the mean SD results and are representative of 3 independent experiments. P 0.05 versus control. NS no stimulation.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Reverse Transcription, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Control, Knock-Out

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 2. TSLP-induced expression of monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), and macrophage colony-stimulating factor (M-CSF) in mouse intervertebral disc cells. A, Mouse intervertebral disc culture lysates were subjected to Western blot analysis with antibodies specific for TSLPR and -actin. Mouse TSLPR-Fc chimera protein (mTSLP-R) was used as a positive control, and human TSLPR-Fc chimera protein (hTSLP-R) was used as a negative control. B, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TSLP for 72 hours. The supernatants were collected and subjected to a cytokine protein array. The table shows the corresponding cytokines on the protein array membrane. C, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TSLP for 72 hours. Supernatants were collected, and MCP-1, MIP-1, M-CSF, and RANTES concentrations were measured by ELISA. Values are the mean and SD. D, For immunohistochemical examination of mouse intervertebral disc tissue, tissue sections were stained with anti-mouse MCP-1 antibody, as described in Materials and Methods. No positive immunohistologic cell staining following stimulation with TNF was seen in control IgG-treated sections (goat-Ab). Results are representative of 3 independent experiments. P 0.05 versus control. GM-CSF granulocyte–macrophage CSF; SDF-1 stromal cell–derived factor 1; IFN- interferon-; TCA-3 T cell–attracting chemokine 3; I-TAC interferon-inducible T cell chemoattractant; TECK thymus-expressed chemokine; KC cytokine-induced neutrophil chemoattractant; TIMP-1 tissue inhibitor of metalloproteinases 1; LIX lipopolysaccharide-induced CXC chemokine; BLC B lymphocyte chemoattractant; sTNF R1 soluble TNF receptor I; GCSF granulocyte CSF (see Figure 1 for other definitions).

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Positive Control, Negative Control, Cell Culture, Protein Array, Membrane, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Control, Derivative Assay

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 3. TSLP induction of MCP-1 production in mouse intervertebral disc cells primarily via the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. A, Intervertebral discs obtained from wild-type mice were cultured in the presence or absence of 10 ng/ml TSLP and/or 10 M SP600125 (JNK inhibitor), 1 M SB203580 (p38 inhibitor), and 10 M LY294002 (PI 3-kinase inhibitor) for 72 hours. Culture supernatants were collected, and the TSLP concentrations were measured by ELISA. B, Intervertebral discs obtained from wild-type mice were cultured in the presence or absence of 10 ng/ml TSLP with or without 10 M LY294002 (PI 3-kinase inhibitor) for the indicated periods of time. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. C, Intervertebral discs obtained from wild-type mice were cultured in the presence of 10 M LY294002 for 72 hours. After washing and replacing the culture medium without LY294002, the discs were then cultured in the presence or absence of 10 ng/ml TSLP for 72 hours. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. D, Mouse intervertebral disc cells were stimulated with 10 ng/ml TSLP for the indicated periods of time (top) and were also stimulated with 10 ng/ml TSLP in the presence or absence of the indicated doses of LY294002 for 60 minutes (bottom). Culture lysates were subjected to Western blot analysis with antibodies specific for phosphorylated Akt. The same membrane was used for Western blot analysis with nonphosphorylated Akt antibody after stripping of the phosphorylated Akt antibody (top and bottom). Results in A–C are the mean and SD and are representative of 3 independent experiments. P 0.05. See Figure 1 for other definitions.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Membrane, Stripping Membranes

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 4. Induction of macrophage migration via monocyte chemotactic protein 1 (MCP-1) by TSLP-activated intervertebral disc culture supernatants. Macrophages (1.5 106) in the upper chamber of a tissue culture insert were allowed to migrate toward the lower chamber, where 3 discs per 96-well plate had been cultured in 1 ml of Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum in the presence or absence of 10 ng/ml TNF or TSLP with or without 10 g/ml anti–MCP-1 antibody for 24 hours. After the incubation of macrophages and TSLP- or TNF- activated disc cultures for 6 hours at 37°C, the membrane was removed, and nonmigrated cells on the upper surface of the membrane were removed. Cells were fixed and stained with Giemsa solution. Cells on the lower surface of the membrane were counted in 4 high-power fields. A, Representative photomicrographs of migrated macrophages on the lower surface of the membrane. B, Quantitative analysis of the number of macrophages that migrated to the lower surface of the membrane. Results are the mean and SD and are representative of 3 independent experiments. P 0.05. See Figure 1 for other definitions.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Migration, Cell Culture, Modification, Incubation, Membrane, Staining

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 5. Partial mediation of TNF-induced monocyte chemotactic protein 1 (MCP-1) production in mouse intervertebral disc cells by TSLP. Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TNF for 72 hours, with or without 10 g/ml mouse or human TSLPR-Fc chimera (mTSLP-R/FC and hTSLP-R/Fc, respectively), or 10 g/ml anti-mouse TSLP antibody or anti-human TSLP antibody. Culture supernatants were collected, and MCP-1 concentrations were measured by ELISA. Results are the mean and SD and are representative of 3 independent experiments. P 0.05. See Figure 1 for other definitions.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 6. Thymic stromal lymphopoietin (TSLP) and monocyte chemotactic protein 1 (MCP-1) expression in herniated disc tissue. Ten herniated disc tissue specimens obtained from 10 patients who underwent surgical resections for a herniated disc were immunohistochemically stained with anti-TSLP and anti–MCP-1 antibodies. No positive immunohistologic cell staining was seen in control IgG-treated sections. A, Representative photomicrographs from one herniated disc specimen. Positive staining is indicated as a brown color. B, Quantitative analysis of the number of hematoxylin-staining cells and TSLP- or MCP-1–immunoreactive cells in 4 high-power fields (100) of herniated disc tissue (n 10). Values are the mean SD. Positive immunoreactive; negative no staining.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Staining, Control

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 1. Physical interactions between TRF2 and BER proteins. A, increasing concentrations of various proteins were spotted in replicate (0.6, 0.8, 0.9, and 1 ng) on a grid of the Discover-Light Protein Array membrane and then hybridized with TRF2 protein (10 ng/mL). After washing, bound TRF2 protein was detected by Western blotting with an anti-TRF2 antibody. B, coimmunoprecipitation of TRF2 and Pol h. HeLa whole-cell extracts (500 ng) were immunoprecipitated with either rabbit anti-TRF2 (lane 3) or control IgG (lane 4) antibodies. The immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti–Pol h or anti-TRF2 antibodies as indicated. TRF2 (lane 1) and Pol h (lane 6) were loaded as markers and positive controls. Input, 10% loaded (lane 2). C, coimmunoprecipitation of TRF2 and FEN-1. HeLa whole-cell extracts (500 ng) were immunoprecipitated with either rabbit anti-TRF2 (lane 3) or control IgG antibodies (lane 1). The immunoprecipitates were probed with anti-FEN-1 or anti-TRF2 antibodies as indicated. Input, 10% loaded (lane 2).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Protein Array, Membrane, Western Blot, Immunoprecipitation, Control, SDS Page

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 2. Mapping the sites of Pol h and FEN-1 interactions with TRF2. A, schematic of the known domains and structural motifs of TRF2 and the borders of the various GST-tagged fragments. B, basic NH2 terminus; TRFH, dimerization domain; Myb, Myb-like telomere DNA binding domain. Numbers indicate the amino acid sequence. B, Coomasie staining of the recombinant GST-tagged TRF2 fragments (2 Ag each) used in the binding assay after single-step purification and SDS-PAGE. C, TRF2 domains that interacted with Pol h and FEN-1. HeLa nuclear extracts (400 AL) were incubated with either GST alone (lanes 2 and 9) or GST-tagged TRF2 fragments (lanes 3-6 and 8) that were prebound to glutathione beads. Eluted proteins were separated by SDS-PAGE, transferred to a membrane, and stained with amido black to ensure equal loading of the various TRF2 fragments. The membrane was probed with mouse anti–Pol h or rabbit anti-FEN-1 antibodies.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Binding Assay, Sequencing, Staining, Recombinant, Purification, SDS Page, Incubation, Membrane

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 4. FEN-1 incision activity in the presence of TRF2. A, FEN-1 incision of a 10-nt flap substrate. Reactions contained 120 pmol/L FEN-1 incubated with a 10-nt flap substrate (100 nmol/L) either alone (lane 2) or together with increasing TRF2 concentrations (9, 18, 90, 180, or 900 pmol/L; lanes 3-7, respectively) at 37jC for 10 minutes. The relative percent incision activity was calculated as described in Materials and Methods and normalized to the FEN-1 alone control (lane 2). Values represent the average and SD of at least three independent experiments. B, FEN-1 incision of a telomeric flap substrate. Reactions contained 10 pmol/L FEN-1 incubated with a 15-nt flap substrate harboring telomeric sequence 5V to the flap (10 nmol/L). FEN-1 was incubated alone (lane 2) or together with increasing TRF2 concentrations (100, 300, and 1,000 pmol/L; lanes 3-5) at 37jC for 10 minutes. The relative percent incision activity was calculated as in (A).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Activity Assay, Incubation, Control, Sequencing

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 5. TRF2 specifically stimulates primer extension by Pol h. A, TRF2 effects on Pol h activity. Pol h (0.5 nmol/L) was preincubated with increasing TRF2 amounts (0, 0.5, 1.5, or 3.0 nmol/L; lanes 2-5, respectively) for 5 minutes on ice. Reactions were initiated by adding the nontelomeric mix15/mix34 substrate (25 nmol/L) and were incubated for 15 minutes at 37jC. Reaction products were run on a 20% denaturing polyacrylamide gel and visualized by a Phosphorimager. Lane 1, substrate alone. E, 0.5 nmol/L (lane 6) and 3.0 nmol/L (lane 7) of heat-denatured TRF2 protein. Lanes 8 and 9, TRF2 (0.5 or 3.0 nmol/L, respectively) in the absence of Pol h. B, quantitation of Pol h primer-extension. Percent of total products with the indicated number of nucleotides incorporated was calculated as described in Materials and Methods. Columns, mean from three independent experiments; bars, SD. C, Klenow activity. Increasing Klenow concentrations (lanes 2-7) were incubated with the mix15/mix34 substrate (25 nmol/L) for 15 minutes at 37jC. Lane 1, substrate alone. Products were analyzed as in (A). D, TRF2 affects on Klenow primer extension. Klenow (0.32 nmol/L) was preincubated with increasing TRF2 amounts (0, 0.32, 0.96, 1.92, or 3.84 nmol/L; lanes 2-6, respectively) and Pol h (0.3 nmol/L) was preincubated with 1.8 nmol/L TRF2 (lane 8) for 5 minutes on ice. Reactions were initiated by adding mix15/mix34 substrate (25 nmol/L) and were incubated for 15 minutes at 37jC. Products were analyzed as in (A).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Activity Assay, Incubation, Quantitation Assay

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 6. Comparison of TRF2 stimulation of Pol h on telomeric and nontelomeric template substrates. A, reactions contained Pol h (0.25 nmol/L) alone or together with increasing TRF2 concentrations (0.125, 0.25, 0.75, and 1.5 nmol/L). Reactions were initiated by adding 25 nmol/L substrate with either nontelomeric template sequence (mix15/mix34; lanes 1-6) or telomeric template sequence (mix15/tel34; lanes 7-12) and were incubated for 15 minutes at 37jC, followed by analysis on a 20% denaturing gel. Quantitation and calculation of primer extension products for the nontelomeric (B) or telomeric (C) template substrates was as described in Materials and Methods. Columns, mean from three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Comparison, Sequencing, Incubation, Quantitation Assay

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 7. TRF2 promotion of Pol h primer extension on substrates with TRF2 binding sites. A, reactions contained Pol h (0.25 nmol/L) alone or together with increasing TRF2 concentrations (0.125, 0.25, 0.75, and 1.5 nmol/L). The reactions were initiated by adding 25 nmol/L telomeric substrate (tel21/tel40) and were incubated for 15 minutes at 37jC, followed by analysis on a 20% denaturing gel. E, 1.5 nmol/L (lane 7) heat-denatured TRF2 protein. B, the percent of products with the indicated number of nucleotides incorporated was calculated as described in Materials and Methods. Columns, mean from at least three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Binding Assay, Incubation

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 8. TRF2 stimulates Pol h strand displacement DNA synthesis on a nontelomeric BER substrate. A, schematic of the 34-bp DNA substrate containing an 8-oxo-guanine at position 17 is shown both before and after treatment with OGG1 and APE1. OGG1 removes the 8-oxo-guanine base and APE1 incises the DNA strand 5V to the resulting apurinic/apyrimidinic site. B, the substrate was pretreated with OGG1 (128 nmol/L) for 20 minutes at 37jC. The pretreated DNA (100 nmol/L) was incubated with 3.4 ng/AL APE1 for 25 minutes at 37jC together with increasing Pol h concentrations (0.6, 1.2, 2.5, and 5 nmol/L; lanes 2-5 and 6-9, respectively). The reactions in lanes 6 to 9 also contained increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L, respectively). C, the OGG1-pretreated DNA substrate was incubated with 3.4 ng/AL APE1 in the absence () or presence (+) of Pol h (5 nmol/L; lane 1) and with increasing concentrations of TRF2 (0, 5, 15, and 30 nmol/L; lanes 2-5, respectively). D, quantitation of unreacted substrate (0), short-patch (1), and long-patch (2-6) BER intermediates. E, quantitation of individual long-patch BER intermediates. Reaction products were calculated as a function of total radioactivity as described in Materials and Methods for reactions containing 5 nmol/L Pol h alone (solid columns) or together with 30 nmol/L TRF2 (hatched columns). Columns, mean of two independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: DNA Synthesis, Incubation, Quantitation Assay, Radioactivity

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 9. TRF2 stimulation of Pol h on telomeric BER substrates. A, a schematic of the 39-bp DNA substrate containing an 8-oxo-guanine at position 17 within two tandem telomeric repeats is shown both before and after treatment with OGG1 and APE1. B, the substrate was pretreated with OGG1 (128 nmol/L) for 20 minutes at 37jC. The pretreated DNA (100 nmol/L) was incubated with 3.4 ng/AL APE1 for 25 minutes at 37jC together with increasing Pol h concentrations (0.62, 1.2, 2.5, and 5 nmol/L; lanes 2-5 and 6-9, respectively). The reactions in lanes 6 to 9 also contained increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L, respectively). C, the OGG1-pretreated DNA substrate was incubated with 3.4 ng/AL APE1 in the presence (+) or absence () of Pol h (1.2 nmol/L), TRF2 (7.5 nmol/L), or FEN-1 (30 nmol/L) as indicated. E, heat-inactivated control. D, quantitation of unreacted substrate (0), short-patch (1), and long-patch (2-7) BER intermediates. E, quantitation of individual long-patch BER intermediates. Reaction products were calculated as a function of total radioactivity as described in Materials and Methods for reactions containing 1.2 nmol/L Pol h alone (solid columns) or together with 7.5 nmol/L TRF2 (hatched columns). Columns, mean of three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Incubation, Control, Quantitation Assay, Radioactivity

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 10. TRF2 enhances Pol h extension of the 3V tail of a telomeric D-loop. The telomeric D-loop substrate (25 nmol/L) was incubated with increasing Pol h concentrations alone (0.62, 1.2, 2.5, and 5 nmol/L; lanes 2-5) or together with increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L; lanes 6-9). The reactions were initiated by adding substrate and were incubated for 15 minutes at 37jC. The reaction products were run on a 20% denaturing and were visualized by a Phosphorimager.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Incubation