Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Effect of overexpressing rat and human CRM1 on Rex activity. (A) REF52 cells were transfected with 0.075 μg of pSRαRex along with 0.5 μg of pDM128RxRE and 0.1 μg of pCDMβ-gal in combination with various amounts of pSRαhCRM1 (⧫) or pSRαrCRM1 (▪). The CAT/β-Gal ratios of all samples were calculated. The ratio of the sample transfected with pSRαRex in the absence of either the hCRM1 or rCRM1 expression plasmid was arbitrarily set to 1. (B) CRM1 proteins produced in REF52 cells that were transfected with 0.3 μg of either rCRM1 or hCRM1 expression plasmids (lane 1, pSRα296; lane 2, pSRαhCRM1; lane 3, pSRαrCRM1) were analyzed by Western blotting as described in Materials and Methods.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Produced, Western Blot

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Ability of hCRM1 and rCRM1 to support Rex function in HeLa cells. (A) HeLa cells were transfected with 0.05 μg of pSRαRex, 0.5 μg of pDM128RxRE, and 0.1 μg of pCDMβ-gal together with various amounts of pSRαhCRM1 or pSRαrCRM1. At 8 h posttransfection, the medium was replaced with medium containing LMB at 0.6 or 0.8 nM. At 24 h posttransfection, the cells were subjected to CAT and β-Gal measurement. The CAT/β-Gal ratios of all samples were calculated. The ratio of the control sample transfected with pSRαRex in the absence of either hCRM1 or rCRM1 expression plasmids and in the absence of LMB was arbitrarily set to 1. The amounts of CAT and the β-Gal activity in the control sample were over 300 pg and 2.5 × 10−3 U, respectively. (B) CRM1 proteins produced in HeLa cells transfected with 0.3 μg of each CRM1 expression plasmid (lane 1, pSRα296; lane 2, pSRαhCRM1; lane 3, pSRαrCRM1) were analyzed by Western blotting.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Expressing, Activity Assay, Produced, Plasmid Preparation, Western Blot

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Interaction of Rex with either hCRM1 or rCRM1. (A) REF52 cells were transfected with the plasmid expressing either GAL-hCRM1 or GAL-rCRM1 in combination with pRexVP, pG5BLuc, and pCDMβ-gal. The Luc/β-Gal ratios of all samples were calculated. GAL-, plasmid expressing only the GAL4 region, which was used as a negative control. (B) Western blot of GAL-hCRM1 and GAL-rCRM1 proteins synthesized in transfected REF52 cells. (C) Pull-down assay using recombinant Rex protein. Purified His-Rex(g10) proteins immobilized on chelating Sepharose were incubated with HeLa or REF52 cell extract in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of GTP-charged recombinant RanQ69L protein. The sample of lane 1 contained neither cell lysate nor recombinant RanQ69L proteins during incubation. The volume of the cell extract subjected to the binding reaction was nine times of that of the input fraction.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Plasmid Preparation, Expressing, Negative Control, Western Blot, Synthesized, Pull Down Assay, Recombinant, Purification, Incubation, Binding Assay

Journal:

Article Title: Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

doi: 10.1128/JVI.75.23.11515-11525.2001

Figure Lengend Snippet: Rex-Rex dimerization analyzed by the coimmunoprecipitation method. HeLa and REF52 cells were transfected with the plasmids which express GAL-Rex and Rex-VP or their derivatives as indicated. At 48 h posttransfection, the cells were harvested and the supernatants were incubated with anti-VP antibodies immobilized on protein-G Sepharose in the presence of GTP-charged recombinant RanQ69L. The recombinant hCRM1 protein was added to the sample, which was derived from the REF52 cells transfected with GAL-Rex and Rex-VP (lane 6) during incubation. The volume of the cell extract subjected to the coimmunoprecipitation reaction was nine times of that of input fraction. Rabbit anti-Rex C terminus antibodies were used for Western blotting.

Article Snippet: A mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and a rabbit anti-Rex C terminus antibody ( 24 ) were used as primary antibodies to detect GAL-fused proteins and the Rex protein, respectively.

Techniques: Transfection, Incubation, Recombinant, Derivative Assay, Western Blot

Journal: Cell

Article Title: Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease

doi: 10.1016/j.cell.2018.08.067

Figure Lengend Snippet:

Article Snippet: Slides were washed in PBS containing 0.05% Tween 20 (PBS-T) and incubated with ImmPRESS HRP Anti-Rabbit or Anti-Mouse IgG (Peroxidase) Polymer Detection Kit (Vector Laboratories, Peterborough, U.K) for thirty minutes at room temperature.

Techniques: Recombinant, Coagulation, Antibody Labeling, Plasmid Preparation, Blocking Assay, Staining, Imaging, Software, Hybridization

Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 1 S100A9 promotes prostate cancer cell invasion and β1 integrin expression through interaction with TLR4. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (A) PC-3 and DU-145 cells invasion was measured by transwell invasion assay. (B) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (C, D) Cell extracts were immunoprecipitated (IP) with control mouse IgG, mouse anti-S100A9 antibody. Immunoblot (IB) was used to detect S100A9, TLR4 and RAGE. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA. (E) TLR4 expression was examined by Western blot after 48 h siRNA transfection. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA followed by stimulation with S100A9. (F) Tumor cell invasion was measured by transwell invasion assay. (G) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Transwell Invasion Assay, Western Blot, Immunoprecipitation, Control, Transfection

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 2 NF-κB mediates S100A9-induced prostate cancer cell β1 integrin up-regulation. PC-3 and DU-145 cells transfected with or without TLR4 siRNA or control siRNA, were transfected with NF-κB-luciferase reporter plasmid, and treated with S100A9 (20 µg/ml) for 48 h. (A, B) Activity of NF-κB was detected by measuring the relative activity of luciferase. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ ml) for 48 h. (C) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Western Blot

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 3 S100A9 promotes prostate cancer cell invasion via integrin β1/FAK signaling. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (A) Fibronectin expression was determined by Western blot. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 48 h. (B) Supernatant fibronectin (FN) concentration was determined by ELISA. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 30 min. (C) The phosphorylation of FAK was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 30 min, the expression of integrin β1 (D) or phosphorylation of FAK (E) was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (F) The invasion activity were measured by transwell invasion assay. (G) PC-3 and DU- 145 cells were treated with β1 integrin functional blocking antibody MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 30 min and the phosphorylation of FAK was measured by Western blot. (H) PC-3 and DU-145 cells were treated with MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 48 h for invasion. (I) PC-3 and DU-145 cells were pretreated for 30 min with FAK inhibitor, PF562271 (100 nM) followed by stimulation with S100A9 (20 µg/ml) for 48 h for invasion. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Transfection, Control, Activity Assay, Transwell Invasion Assay, Functional Assay, Blocking Assay

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 4 S100A9 induces prostate cancer cell metastasis in vivo. DU-145 cells were transfected with pcDNA3.1 or pc DNA-S100A9 plasmid. (A, B) The expression or secretion of S100A9 was determined by Western blot and ELISA. The cells were injected to nude mice via tail vein. 30 days after inoculation, nude mice were sacrificed. (C) Expressions of S100A9 and integrin β1 in xenograft tumors were detected by immunohistochemistry. (D) Expressions of FAK, p-FAK, NF-kB p65 and p-NF-kB p65 in xenograft tumors were determined by Western blot. (E) Micrometastatic tumors in the lungs of mouse xenografts were counted and subjected to H and E staining. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M.*p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Immunohistochemistry, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Integrated multiomic analysis identifies TRIP13 as a mediator of alveolar epithelial type II cell dysfunction in idiopathic pulmonary fibrosis

doi: 10.1016/j.bbadis.2024.167572

Figure Lengend Snippet: Key resources.

Article Snippet: Mouse IgG1 anti-human CD326 (EpCAM)-conjugated microbeads , Miltenyi Biotec , Cat# 130–061–101 RRID: AB_2832928.

Techniques: Plasmid Preparation, Magnetic Beads, Recombinant, Blocking Assay, Lysis, Protease Inhibitor, Red Blood Cell Lysis, Software, Microscopy, Fluorescence, Imaging