Antibody data
- Antibody Data
- Antigen structure
- References [47]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [3]
- Other assay [26]
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Validation data
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- Product number
- MA1-140 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- beta Actin Monoclonal Antibody (15G5A11/E2)
- Antibody type
- Monoclonal
- Antigen
- Purifed from natural sources
- Description
- By Western blot, MA1-140 detects a prominent ~42kD protein in human, mouse, non-human primate, and rat whole cell lysates. For visualization of actin filaments by immunofluorescence, fixation and permeabilization with methanol is required.
- Reactivity
- Human, Mouse, Rat, Canine
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- 15G5A11/E2
- Vial size
- 100 µg
- Concentration
- 1 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide.
The phosphatase and tensin homolog gene inserted between NP and P gene of recombinant New castle disease virus oncolytic effect test to glioblastoma cell and xenograft mouse model.
Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome.
Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells.
A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.
miR-377 inhibition enhances the survival of trophoblast cells via upregulation of FNDC5 in gestational diabetes mellitus.
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Protective effects of gastrodin pretreatment on mouse hepatic ischemia-reperfusion occurring through antioxidant and anti-apoptotic mechanisms.
Fibronectin assembly regulates lumen formation in breast acini.
SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.
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α-synuclein aggregates induce c-Abl activation and dopaminergic neuronal loss by a feed-forward redox stress mechanism.
Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer.
Liposomes Loaded with Everolimus and Coated with Hyaluronic Acid: A Promising Approach for Lung Fibrosis.
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Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis.
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Systemic Delivery of AAV-Fdxr Mitigates the Phenotypes of Mitochondrial Disorders in Fdxr Mutant Mice.
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Adverse Effects of Coumestrol and Genistein on Mammary Morphogenesis and Future Milk Production Ability of Mammary Epithelial Cells.
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Preventive Effects of Different Fermentation Times of Shuidouchi on Diphenoxylate-Induced Constipation in Mice.
An ANGPTL4-ceramide-protein kinase Cζ axis mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia in mice.
Characterization of polyclonal antibodies to Herpes Simplex Virus types 1 and 2.
CRISPR/Cas9‑mediated hypoxia inducible factor‑1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma.
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Radiation-induced DNA damage and repair effects on 3D genome organization.
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American journal of physiology. Gastrointestinal and liver physiology 2019 Nov 1;317(5):G682-G693
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The Journal of biological chemistry 2019 Jun 7;294(23):9213-9224
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Lloyd J, Copaciu R, Yahyabeik A, DeWit C, Cummings K, Lacey M, Su Q
Journal of histotechnology 2019 Dec;42(4):202-214
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Molecules (Basel, Switzerland) 2018 May 3;23(5)
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Oncology reports 2018 Feb;39(2):483-490
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Proteomic analysis of exosomes derived from human lymphoma cells.
Yao Y, Wei W, Sun J, Chen L, Deng X, Ma L, Hao S
European journal of medical research 2015 Jan 29;20(1):8
European journal of medical research 2015 Jan 29;20(1):8
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of beta-actin was performed by loading 25 µg of various cell lysates and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543). The membrane was probed with a beta-actin monoclonal antibody (Product # MA1-140) at a dilution of 1:16,000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:40,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: Similar results are obtained when blocking with 5% BSA in TBST.
Supportive validation
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- Invitrogen Antibodies (provider)
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- Immunofluorescent analysis of beta-actin (green) in HeLa cells. Cells were fixed and permeabilized with ice-cold methanol for 10 minutes at room temperature, and blocked with 0.3% BSA in PBS for 15 minutes at room temperature. Cells were probed with a beta-actin monoclonal antibody (Product # MA1-140) at a dilution of 1:2000, or incubated in blocking buffer as a negative control, overnight at 4°C, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:1000 for 30 minutes at room temperature. Nuclei (blue) were stained with DAPI (Product # 62248). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
- Submitted by
- Invitrogen Antibodies (provider)
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- Immunofluorescent analysis of Beta- Actin (green) in U2OS cells untreated or treated with 1uM Actinomycin D for 20 hours. The cells were fixed with 4% Paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a Beta-Actin mouse monoclonal antibody (Product # MA1-140) at a dilution of 1:2000 in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32723) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of Beta- Actin (green) in MDCK cells. The cells were fixed with 4% Paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a Beta-Actin mouse monoclonal antibody (Product # MA1-140) at a dilution of 1:1000 in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32723) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunoprecipitation of beta-actin was performed using NIH/3T3 cells. Antigen-antibody complexes were formed by incubating 500 µg of NIH/3T3 whole cell lysates with 2 µg of a beta-actin monoclonal antibody (Product # MA1-140) overnight on a rocking platform at 4°C. The immune complexes were captured on 100 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). The IP eluate (right lane) and 15 µg of NIH/3T3 lysate (left lane) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with a beta-actin monoclonal antibody (Product # MA1-140) at a dilution of 1:8000 overnight rotating at 4°C, washed in TBST, and probed with n HRP-conjugated anti-mouse light chain secondary antibody a dilution of 1:40,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Figure 2. Generation of the CRISPR/Cas9-based lentivirus and HIF-1 alpha knockout in SMMC-7721 cells and in the SMMC-7721-induced tumor tissues of mice. (A) Immunohistochemical analysis for the detection of HIF-1alpha expression in hepatocellular carcinoma tissues following infection with LV-Ctrl or LV-H721 in the subcutaneous animal model (bars, 100 um). (B) Diagram illustration of the lentiviral vector ( Lenti-CAS9-sgRNA-egfp ). Three sgRNAs (sgRNA719, sgRNA720 and sgRNA721) targeting HIF-1alpha were designed, and Lenti-CAS9-sgRNA719 / 720 /721 plasmids were constructed. (C) Gel electrophoresis and DNA analysis of the HIF-1alpha genomic frame shift mutation conducted after the T7E1 endonuclease assay in SMMC-7721 cells infected with LV-Ctrl or LV-H719/720/721. (D) Western blot analysis of HIF-1alpha expression in the different lentivirus-infected SMMC-7721 cells with CoCl 2 -simulated hypoxia. (E) GFP-positive cells analyzed by flow cytometry following infection with LV-Ctrl and LV-H721. (F) Relative mRNA and (G) protein expression levels of VEGF and MDR1 in different cell groups were examined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. beta-actin served as an internal control. Data are a representation of four repeated experiments, and histograms represent the mean +- standard deviation. ***P
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- Figure 1 Cytotoxic activity of GNP-HCIm and Imatinib (Im) alone on BOS-derived LFs. ( A ) Cell viability assay after 24, 48 and 72 h of treatment with GNP-HCIm and Im alone at the same concentration (10 muM). Data of two independent replicates (N = 10 for each condition) are represented as mean +- SD. ( B ) Quantification of apoptotic and necrotic cells after 48 h of treatment with GNP-HCIm and Im alone. Apoptotic cells were labeled with PE-Annexin V and necrotic cells with 7-AAD. Data of three independent replicates (N = 9 for each condition) are represented as mean +- SD. ( C ) Semiquantitative analysis of immunoblot of cAbl activity in BOS-derived cells after treatment with GNP-HCIm and Im alone for 24 h. Activity of cAbl was assessed by the quantification of phosphorylated protein related to total cAbl (cAbl-p/cAbl) normalizing results obtained after treatments with CTR cells. ( D ) representative immunoblot using antibodies specific for cAbl-p, or c-Abl or beta-actin. Line 1 = CTR; line 2 = GNP-HCIm; line 3 = Im alone. Data of two independent replicates (N = 6 for each condition) are represented as mean +- SD. All graphs are made by Graphpad Prism 6.0; ( https://www.graphpad.com/scientific-software/prism/ ). All data were analyzed by one-way ANOVA followed by a Tukey post-hoc test for multiple comparisons. ***, p < 0.001 vs. CTR; **, p < 0.01 vs. CTR; ^, p < 0.001 vs. Im.
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- FIGURE 1 Functional consequences of Orai1 protein level manipulation by in vivo plasmid electroporation. (A) Good efficiency of venus-Orai1 expression in Cmpt mice where a high percentage of the muscle fibers were transfected as illustrated by them displaying bright fluorescence signals. (B) Representative image showing correct targeting of venus-Orai1 to the triad in Cmpt mice as highlighted by the double-banded pattern of venus' fluorescence. The white trace illustrates the fluorescence intensity profile plotted as a function of distance along the white line for five successive triads. (C) Immunoblot analysis in WT mice 5-days after the small-hairpin RNA (shRNA) against Orai1 was introduced into FDB muscles using electroporation showed a significant reduction in the Orai1 protein levels. A scrambled non targeted shRNA probe was used as control along with samples collected from the untreated WT mice. The analysis was performed on whole FDB muscles and also using ~30 individual FDB fibers identified via the GFP signal and manually collected using a fluorescence microscope. Actin was used as internal control. (D) Quantitative densitometry analysis of four independent immunoblots revealed a significant decrease of Orai1 levels compared to WT taken as 100% in both whole FDB muscles and single fibers, respectively ( # p < 0.001) (E) A representative SOCE measurement showing the fluorescence profile versus elapsed time following various solution exchanges performed on a Cmpt fiber
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- Figure 1 Establishment of SARS-CoV-2 S1-NanoBiT biosensor . (A) Schematic of SARS-CoV-2 viral entry with Spike (S) protein interaction with cellular receptor, ACE2. (B) Overall design of Nanoluciferase complementation-based biosensor of SARS-CoV-2 Spike S1 and ACE2 ectodomain interaction. (C) Immunoblot analysis of S1-LgBiT and SmBiT-ACE2 expression in SARS-CoV-2 S1 NanoBiT construct transfected HEK293T cells. beta-actin and total protein loading are shown as controls. (D) Demonstration of the complementation-based nature of the designed S1-LgBiT and SmBiT-ACE2 interaction nano-luciferase complementation-based biosensor. Figure 1
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- Fig 4 JEV and WNV inhibit collagen, PAM and SSBP2 expression in a neuron model. SK-N-SH cells were infected with JEV or WNV at a MOI of 1 and proteins were extracted for western blot analysis 48hpi. Two independent experiments are displayed on the blot.
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- Fig. 2 C212 induces leukemia cell apoptosis. Growing leukemia cells K562 and HL60 were treated with C212 at the indicated doses for 24 h and subjected to apoptosis assay with Annexin-V & PI staining ( a ), mitochondrial membrane potential MMP assay with JC-1 staining ( b ), and Western blot of apoptosis marker proteins (in K562, c ; in HL60, d )
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- Fig. 6 C212 decreases Hsp90 client protein levels and induces protein aggregates. a Concentration-dependent quenching effect of C212 (0-50 muM) on the intrinsic fluorescence of N-, M-, and C-domains of Hsp90. Y- and X-axis indicate fluorescence intensity and emission wavelength, respectively. Each curve represents the average of triplicate measurements. b Leukemia cells (HL60, left; K562, right) were treated with C212 at the indicated doses for 24 h, followed by Western blot of Hsp90 client proteins in cell lysates. c HL60 (top) and K562 (bottom) cells that were serum-starved as in Fig. 4 a and b to induce quiescence/slow-growth were further treated with C212 at the indicated doses for 30 and 36 h, respectively, then subjected to fluorescence-based protein aggregation assay using ProteoStat. Error bar, SEM (n = 2); * p < 0.05, ** p < 0.01 (over control at 0 muM). d Quiescent HL60 (top) and K562 (bottom) cells were treated with C212 at the indicated doses for 30 and 36 h, respectively, in the presence or absence of 12 muM CQ, then subjected to fluorescence-based protein aggregation assay using ProteoStat. Error bar, SEM (n = 2); * p < 0.05, ** p < 0.01, *** p < 0.001. e HL60 (left) and K562 (right) cells were serum-starved, treated with C212 at the indicated doses in the presence or absence of CQ, and serum-stimulated as in Fig. 5 a and b, respectively. Cells were then harvested and subjected to Click-iT EdU assay. f A proposed model of C212 to deepen quiescence of leukemia ce
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- Fig. 7 Accumulation of autophagosomes and lysosomes in the Atp6v0a1 A512P/A512P brain. a , b Immunostaining of the autophagosome maker LC3 and the lysosomal membrane marker Lamp2 in coronal sections of the cortex ( a ) and the hippocampal CA3 region ( b ) of Atp6v0a1 +/+ (WT) and Atp6v0a1 A512P/A512P (Homo) pups at P10. LC3 was accumulated in the cell bodies (arrowheads in a ) and neurites (arrows in a ) in layer V of the cortex, and in hippocampal mossy fibers (arrows in b ) and in the cell bodies of CA3 neurons (arrowheads in b ), in which expanded Lamp2 distributions were also noticeable. In the bright field images in b , healthy CA3 neuronal cells displayed as round-shaped cells with translucent cytoplasm, but abnormal cells with a black boundary were also observed. Scale bars, 50 mum. c Immunoblot analysis of LC3 levels in the cortex (CTX), hippocampus (Hip), and cerebellum (CB) of Atp6v0a1 +/+ and Atp6v0a1 A512P/A512P pups at P10. d , e Quantification of the band intensity of the immunoblot. The autophagosomal membrane-bound form of LC3, LC3-II, was increased in CTX of Atp6v0a1 A512P/A512P pups. LC3-II intensity was normalized to the LC3-I ( d ) and beta-actin ( e ) intensity, and ratios to the average intensity of WT pups were shown. Data represented as mean values +- standard deviation of three independent experiments. Two way ANOVA with Sidak''s multiple comparisons test was used. f Electron microscopy images in hippocampal CA3 region of Atp6v0a1 +/+ and Atp6v0a1 A51
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- Figure 7 Western blot analysis of mTOR and p-mTOR on LFs derived from ( a ) CLAD and ( b ) CTD-ILD after 24 h of treatment with liposomes and everolimus alone (50 nM). ( a , b ) Representative blot of immunedecoration using anti-mTOR, anti-p-mTOR, and beta-actin. ( c ) Quantitative analysis of immunoblots representing the expression level of p-mTOR in CLAD and CTD-ILD normalized to CTR = 1.
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- Figure 3 Changes of pre-RNA processing machinery after 4 days of Mettl3 KD. ( A ) Quantification of RT-qPCR data (* p
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- Figure 1. Chemical and genetic ablation of ATR signaling depletes the abundance of key resection factors. ( A ) U-2OS cells were cultured for 5 days in medium containing DMSO or the indicated concentrations of ATRi VE-821 and analyzed by immunoblotting. ( B ) Quantification of blots in ( A ). ( C ) U-2OS cells were treated as in ( A ) but with the ATRi AZD6738. ( D ) Quantification of blots in ( C ). ( E ) IdU incorporation analysis of U-2OS cells treated as in ( C ). ( F ) Strategy for abrogating ATR activators using the HCT116- ETAA1DeltaAAD -TOPBP1-mAID cell line. ( G ) Immunoblot analysis in HCT116- ETAA1DeltaAAD- TOPBP1-mAID cells after 2 days in auxin. ( H ) Immunoblot analysis of HCT116- ETAA1DeltaAAD- TOPBP1-mAID cells treated for 2 days with auxin and released in fresh medium for additional 2 days. ( I ) IdU incorporation analysis of HCT116- ETAA1DeltaAAD -TOPBP1-mAID cells treated as in ( G ).
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- FIGURE 1 Protein phosphatase 2A (PP2A) specifically dephosphorylates cytoplasmic ATR at S428 and its action involves direct binding to ATR. (A) A549 cells were treated with siRNAs against three protein phosphatases (PP2Ac, PP4c, and PP5c) followed by UV treatment at 40 J/m 2 with a 2 h recovery. Analysis of cytoplasmic extracts reveals that PP2A is required to dephosphorylate cytoplasmic pATR (S428). (B) siRNA knockdown of PP2Ac in A549 cells was followed by UV treatment at 40 J/m 2 with a 2 h recovery. pATR (S428) levels are increased in cytoplasm with PP2Ac knockdown while the phosphorylation status of ATR in the nucleus remains unchanged. WB of whole cell extracts shows PP2A siRNA knockdown efficiency in A549 cells (C) HCT 116 ATR flox/- cells were transfected with N-terminal Flag-tagged wild-type (WT) or S428A mutant ATR expression constructs and UV treated at 40 J/m 2 with a 2 h recovery. Analysis of the cytoplasmic extracts reveals that PP2A specifically targets the pATR (S428) residue. The pATR (S428) observed in the S428A cells reflects some phosphorylation of the residual endogenous ATR remaining in the ATR flox/- cells (right panel). (D) PLA revealing that in A549 cells there is a direct interaction between ATR and PP2A, especially after UV irradiation at 40 J/m 2 . A DAPI-staining overlay is used to show location of the nuclei. The bar graph represents a statistical analysis of the PLA images. (E) A549 cells treated with UV at 40 J/m 2 were lysed, followed by co-im
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- FIGURE 2 MSC Exosome mediated delivery of Zinc Finger Protein Activator increases the expression of CFTR. (A) TEM micrograph of exosomes isolated from the culture medium of MSC-CFZF-VPR transfected cells. Exosomes were measured by using Nanosight NS 300 system in the supernatant from cultures cells. The histogram represents particle size distribution. (B) Western blot analysis for exosome markers in CFZF-VPR transfected MSC (CFZF) and non-transfected MSCs (Neg) derived exosomes. (C) Western blot of FLAG-tagged CFZF-VPR protein enriched in exosomes from left: MSC-CFZF-VPR or right: non-transfected MSC-exosomes (M-Ex), HEK293-exosomes (H-Ex) and corresponding MSC cell lysate (M-CL) samples, respectively. The Image J values from the Flag-tag (Flag) relative to Beta Actin (B-Act) are shown below the blot. (D) Evaluation by qRT-PCR of mRNA expression of exosomes from CFZF-VPR/Cx43-transfected MSCs. The results from triplicate exosomes collected from three different CFZF-VPR/Cx43 transfected MSCs are shown. (E) Light microscopy immunofluorescence images of HuBECs uptake of MSCs-CFZF-VPR exosomes labelled with BODIPY TR ceramide (red), Nuclei (Blue), Actin (Green). Scale bar, 10 mum. (F) CFTR expression was determined by qRT-PCR following treatment with MSC exosomes directed to the CFTR promoter (MSC-CFZF-VPR) or Control (MSC-Neg) in HuBECs. For E and F experiments were performed in triplicate with 10e+03 HuBECs treated with 5e+10 exosomes. Experiment shown the standard error of the
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- Figure 4 miR-377-3p negatively regulated FNDC5 expression. (a) The predicted seed-recognition sites in the 3'-UTR of FNDC5 mRNA and the miR-377-3p sequences were shown. (b) Relative luciferase activity of the FNDC5 3'-UTR reporter plasmid in 293 T and HTR-8/SVneo cells after transfection with NC mimic or miR-377-3p mimic. The mutant FNDC5 3'-UTR reporter was also used as a control. (c) Protein expression levels of FNDC5 in HTR-8/SVneo and BeWo cells from groups of control, HG, HG + NC inhibitor, HG + miR-377-3p inhibitor, HG + NC mimic, HG + miR-377-3p mimic, as determined using western blotting. Bands were quantified and shown in histogram. The data are expressed as mean +- SEM. ** indicates p < 0.01.
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- Figure 1 Fibril formation decreases in hypoxia-exposed breast epithelial cells. (A) MCF10A, MCF10AT and MCF10 DCIS.com cells were exposed to hypoxia (1% O 2 ) for the indicated times and cell lysates fractionated using deoxycholate to separate fibril FN and soluble FN. (B) Ratio of fibril versus soluble FN fractions, normalized to loading control GAPDH is plotted as mean +- SEM . Quantification is an average of three independent trials. Statistically significant differences between untreated and hypoxic samples were calculated using unpaired Student's t -test. Actual p values are included. (C) Total cell lysates at the indicated treatment times were lysed in sodium dodecyl sulfate buffer to solubilize total FN pools (fibril and soluble combined) and immunoblotted against FN. Vinculin is used as loading control. (D) Total FN levels normalized to vinculin are plotted as mean +- SEM of three independent trials. Statistical significance and actual p values were determined using the unpaired Student t -test. (E) Total cell lysates immunoblotted against proteins as indicated. Actin was used as loading control
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- Figure 10 Role of HKTs on the immunosuppressive (TGFbeta and CD47) and stem-like cell (CD133) markers of TAM-activated ( A ) and TAF-activated ( B ) B16F10 melanoma cells. TGF-beta concentration was analyzed with ELISA, while CD47 and CD133 were analyzed with WB. Bands for CD47, CD133, and beta-actin proteins were visualized by enhanced chemiluminescence (ECL Plus) and quantified relative to beta-actin using densitometry and Molecular Analyst Software. Uncropped blots of CD47, CD133, and beta-actin proteins are provided in the supplementary materials (Figure S1A-C, respectively). * p< 0.05; ***** p< 0.001; ** p< 0.0001.
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- Fig. 2 CADA downmodulates cell-surface huCD4 and PTK7 in lymphoid cells and the transient expression of huCD4, SORT, PTK7, ERLEC1, and DNAJC3 in transfected cells. A , concentration-response curves of CADA for huCD4 and PTK7 in SUP-T1 cells. Cells were incubated with CADA for 24 h. Tested CADA concentrations were 50, 10, 2, 0.4, 0.08, and 0.016 muM. Expression of the surface receptors was measured with flow cytometry and normalized to the DMSO control. A four parameter concentration response curve was fitted to data from at least three replicate experiments. Values are mean +- SD; n >= 3. B , Western blot images of cell lysates from nontransfected (NT) and huCD4-V5, SORT-V5, PTK7-V5, ERLEC1-V5, or DNAJC3-V5 transfected HEK293T cells treated for 24 h with different CADA concentrations. Protein bands were visualized using an antibody against the V5 tag, whereas for the cell loading control, an anticlathrin or anti-beta-actin antibody was used. One representative experiment out of 3 to 5 is shown. C , concentration-response curves of CADA for huCD4, SORT, PTK7, ERLEC1, and DNAJC3 in transfected HEK293T cells. Samples from ( B ) were quantified and normalized to the clathrin (huCD4, ERLEC1, and DNAJC3) or beta-actin (SORT and PTK7) internal control. A four parameter concentration response curve was fitted to the data from at least three replicate experiments. Values are mean +- SD; n >= 3. CADA, cyclotriazadisulfonamide; DMSO, dimethyl sulfoxide; DNAJC3, DnaJ homolog subfamily C
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- Figure 2 CCK receptor inhibition with proglumide decreases fibrosis in the pancreatic TME. ( A ) Representative images from each treatment group of tumors from the mT3-2D subcutaneous murine pancreatic cancer reacted with Masson's trichrome stain to demonstrate fibrosis in the pancreatic tumor microenvironment. Images shown are at magnification 10x (bar = 200 um). ( B ) Quantitative analysis of fibrosis for the murine syngeneic mT3-2D tumors was determined by ImageJ. Mean +- SEM from n > 50 images. Significantly less fibrosis was detected in the proglumide and combination-treated tumors ( # compared to gemcitabine, * compared to PBS controls; ## p < 0.005, *** p < 0.005, ### p < 0.001). ( C ) Representative images from each treatment group of PANC-1 orthotopic tumors reacted with Masson's trichrome stain to demonstrate fibrosis in the pancreatic tumor microenvironment. Images shown are at magnification 10x. ( D ) Quantitative analysis of fibrosis in PANC-1 tumors was determined by ImageJ. Mean +- SEM from n
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- Figure 4 Combination therapy with gemcitabine and proglumide decreases metastases by preventing epithelial to mesenchymal transition (EMT). Quantitative PCR analysis for mRNA expression of transcription factors and proteins from PDAC tumors ( n = 4 per group) with relative gene expression (2 -DeltaDeltaCt +- SEM) for: ( A ) Vimentin , ( B ) Zeb1 , ( C ) Zeb2 , ( D ) SNAIL1 , ( E ) beta-CATENIN , and ( F ) TBFbetaR2 . Unpaired t-test was used for analysis (* p = 0.05; ** p < 0.01) and GAPDH was used as the loading control. ( G ) Western blot for phospho-paxillin for tumor protein extracts of each group normalized with beta -actin. (The whole western blot is shown in the Figure S4 ). ( H ) Analysis of bands by densitometry show significant differences in the groups * p < 0.05. ( I ) Western blot of PANC-1 tumor protein extracts reacted with an antibody for beta-catenin normalized with beta -actin. (The whole western blot is shown in the Figure S4 ). ( J ) Analysis of the bands by densitometry show a decrease in the intensity of the bands in the proglumide and combination groups although this did not quite reach statistical significance.
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- Fig. 3 Effects of PTEN gene delivered by rNDV to apoptosis suppressor genes of T98G glioblastoma cells. A PTEN, P-Akt, and hTERT protein expression analysis using western blotting of PTEN inserted rNDV Position ""1"" infected glioblastoma cells, PTEN inserted rNDV Position ""2"" infected glioblastoma cells, rNDV infected glioblastoma cells, and not infected glioblastoma cells. B-actin protein western blotting is for loading control. B Relative level of PTEN, P-Akt and hTERT mRNA using Real-Time quantitative PCR in PTEN inserted rNDV Position ""1"" infected glioblastoma cell (T98G), PTEN inserted rNDV Position ""2"" infected glioblastoma cell (T98G), rNDV infected glioblastoma cell (T98G) and not infected glioblastoma cell (T98G) (negative control). Significant differences are indicated as * p < 0.05 and ** p < 0.01. C PTEN protein expression analysis using western blotting of PTEN inserted rNDV Position""1'' infected glioblastoma cell (T98G), rNDV infected glioblastoma cell (T98G), and not infected glioblastoma cell (T98G) (negative control) at 3,6,9 h after infection
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- Figure 6 Proglumide's interaction with FXR. ( A ) Results of proglumide interaction at the FXR as evaluated by the FXR reporter assay shows the expected sigmoidal curve of the FXR agonist, GW4064, with an EC 50 ~312.7 nM. Proglumide reacts with the FXR receptor with a characteristic agonist curve similar to GW4064 and an EC 50 ~214.9 nM. ( B ) Results of the reporter assay showing characteristic plot with the agonist (GW1464) compared to that of the FXR antagonist DY268. ( C-a ) FXR protein expression by Western blot is shown for NASH livers of mice on CDE/Reg diet (a) N = 10) and from livers of mice on CDE/Prog diet (( C-b ), N = 10)). Protein expression is normalized with beta-actin. ( D ) Densitometry analysis of the Western blot above for FXR protein expression is analyzed and plotted as a ratio normalized to beta-actin. FXR expression is significantly increased in the mice on the CDE diet treated with proglumide compared to mice on the CDE diet with untreated water (* p = 0.03). ( E ) FXR mRNA expression as measured by qRT-PCR shows a decrease in the FXR expression in the CDE/Reg-fed mouse livers compared to FXR expression in the normal mouse liver. Restoration of the mRNA levels to control levels is shown in the livers of mice treated with proglumide (* p = 0.042).
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- Figure 2. Antibody validation. (a) Coomassie blue-stained sodium dodecylsulfate (SDS)-polyacrylamide gel and the Western blots immunoprobed with (b) polyclonal anti-HSV-1, (c) monoclonal anti-HSV-1 10A3, (d) polyclonal anti-HSV-2 and (e) monoclonal anti-b-actin. Lane 1, molecular weight markers from 25 to 250 kilodaltons (kD) for (a-d), 55 and 70 kD for (e) with the position of b-actin (42 kD) (arrow); Lanes 2-9, lysates of human cells infected with HSV-1, HSV-2 and other herpes viruses; Lane 10, lysate of non-infected Vero cells. mab, monoclonal antibody; pab, polyclonal antibody.
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- Fig. 1 U87 cells have no detectable endogenous ACE2 expression but high levels of Cathepsin B. A) Different cell lines were analyzed for ACE2 expression by immunoblotting, with beta-actin as loading control. B) Same as in (A) but with the stably ACE2 transduced U87 cells, tested at an early (# 1) or late (# 32) passage of the cells. The transduced U87 cells stably express ACE2 at a very high level as compared to endogenous ACE2 in the Vero E6 cells. The same immunoblot is used for panel A and B, but with a longer exposure time for panel A in order to visualize the faint band in the Calu-3 sample. C) Comparative qPCR analysis of TMPRSS2 mRNA levels in different cells. Graph shows individual copy numbers/mul of 3 technical replicates (n = 3) as calculated from a TMPRSS2 standard. The TMPRSS2 level in Vero E6 cells was below detection limit. D) Different cell lines were analyzed for Cathepsin L (CTSL; left) and Cathepsin B (CTSB; right) expression by immunoblotting, with clathrin as loading control. For CTSL, different species are detected (indicated by the bracket): Pro-CTSL (41 kDa), glycosylated mature CTSL (31 kDa) and non-glycosylated mature CTSL (25 kDa), whereas only the Pro-CTSB form (42 kDa) is visualized. M: molecular marker in kDa, UD: undetectable. Fig. 1