monoclonal anti beta actin antibody (Millipore)
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Name:
Monoclonal Anti beta Actin antibody
Description:
Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three alpha actins skeletal cardiac smooth one beta actin beta non muscle and two gamma actins gamma smooth muscle and gamma non muscle Recognizes an epitope located on the N terminal end of the beta isoform of actin It specifically labels beta actin in a wide variety of tissues and species In immunofluorescent staining of chicken gizzard ultrathin cryosections the antibody labels the dense bodies the longitudinal channels linking consecutive dense bodies that are also occupied by desmin and the membrane associated dense plaque The antibody does not react with adult cardiac or skeletal muscle besides traces due to contamination of the sample with non muscle cells or with beta actin expressing cells in Dictyostelium discoideum amoeba The epitope recognized by the antibody is resistant to formalin fixation and paraffin embedding B5 methacarn ethanol or Bouin s solutions may be also used as fixatives The antibody can be used as probes for beta actin as an internal control in immunoblotting
Catalog Number:
A5316
Molecular Weight:
antigen mol wt 42 kDa
Price:
None
Applications:
Mouse Monoclonal Anti-β-Actin antibody has been used for western blot analyses. The antibody can also be used for immunohistochemistry, indirect immunofluorescence (1:1,000) using cultured human or chicken fibroblasts, and indirect ELISA.Western blot analysis of MDCK cell lysates were performed using monoclonal anti-actin antibody as a primary antibody.Monoclonal mouse anti-actin antibody was used as a loading control for western blot analysis of immunoprecipitated proteins from rat dorsal root ganglion cocultures.
Host:
mouse
Conjugate:
unconjugated
Immunogen:
slightly modified β-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys, conjugated to KLH.
Category:
Antibodies
Isotype:
IgG2a
Source:
goat
Reactivity:
human
Score:
85
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Structured Review
Millipore
monoclonal anti beta actin antibody
Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three alpha actins skeletal cardiac smooth one beta actin beta non muscle and two gamma actins gamma smooth muscle and gamma non muscle Recognizes an epitope located on the N terminal end of the beta isoform of actin It specifically labels beta actin in a wide variety of tissues and species In immunofluorescent staining of chicken gizzard ultrathin cryosections the antibody labels the dense bodies the longitudinal channels linking consecutive dense bodies that are also occupied by desmin and the membrane associated dense plaque The antibody does not react with adult cardiac or skeletal muscle besides traces due to contamination of the sample with non muscle cells or with beta actin expressing cells in Dictyostelium discoideum amoeba The epitope recognized by the antibody is resistant to formalin fixation and paraffin embedding B5 methacarn ethanol or Bouin s solutions may be also used as fixatives The antibody can be used as probes for beta actin as an internal control in immunoblotting
https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
Average 99 stars, based on 1583 article reviews
Price from $9.99 to $1999.99
Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three alpha actins skeletal cardiac smooth one beta actin beta non muscle and two gamma actins gamma smooth muscle and gamma non muscle Recognizes an epitope located on the N terminal end of the beta isoform of actin It specifically labels beta actin in a wide variety of tissues and species In immunofluorescent staining of chicken gizzard ultrathin cryosections the antibody labels the dense bodies the longitudinal channels linking consecutive dense bodies that are also occupied by desmin and the membrane associated dense plaque The antibody does not react with adult cardiac or skeletal muscle besides traces due to contamination of the sample with non muscle cells or with beta actin expressing cells in Dictyostelium discoideum amoeba The epitope recognized by the antibody is resistant to formalin fixation and paraffin embedding B5 methacarn ethanol or Bouin s solutions may be also used as fixatives The antibody can be used as probes for beta actin as an internal control in immunoblotting
https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
Average 99 stars, based on 1583 article reviews
Price from $9.99 to $1999.99
monoclonal anti beta actin antibody - by Bioz Stars,
2019-12
99/100 stars
Related Products / Commonly Used Together
Images
1) Product Images from "PP2A mediates apoptosis or autophagic cell death in multiple myeloma cell lines"
Article Title: PP2A mediates apoptosis or autophagic cell death in multiple myeloma cell lines
Journal: Oncotarget
doi: 10.18632/oncotarget.20415
Figure Legend Snippet: Akt inactivation mediates apoptosis in IM-9 cells (A) IM-9 and IM-9/ Bcl-2 cells were treated with BetA for different periods of time. Treated cells were lysed for detecting Akt phosphorylation (p-Akt), total Akt (t-Akt), Bax oligomerization, conformational change and mitochondrial translocation by Western blotting, with β-Actin serving as a loading control. Bax conformational change was detected as described before (Hu et al., 2012b). (B) IM-9 and IM-9/ Bcl-2 cells were transfected with the constitutively active Akt1 for 48 h, and then treated with BetA for 48 h. treated cells were lysed for Western blotting. (C) Cells were treated with BetA and/or 25 mM LY294002 for 72 h, and then cells were lysed and assayed for individual protein levels by Western blot. (D) Cells were treated with BetA for 48 h. Top row , treated cells were collected for detection of cell viability. Graphs showing results of quantitative analyses ( n =3, mean ± S.D. *, P
Techniques Used: Translocation Assay, Western Blot, Transfection
Figure Legend Snippet: BetA inhibits tumor growth and induces apoptosis or autophagic cell death in IM-9 or IM-9/Bcl-2 xenograft in vivo (A) The cell lines were subcutaneously injected into the right dorsal flank of 6- to 8-week-old female athymic nude BALB/c mice. Following tumor growth for 7 days, the tumor-bearing mice were randomly assigned into the following two groups (10 mice per treatment group): Ctrl group and BetA-treated group. Mice were injected intraperitoneally with BetA as described in the Materials and Methods section. Tumor size was measured at the indicated days and volume was calculated (*, P
Techniques Used: In Vivo, Injection, Mouse Assay
Figure Legend Snippet: PP2A mediates autophagic cell death or apoptosis through regulating its connection with Akt and DAPK (A) Left row , Cells were pre-incubated with indicated concentrations of DEVD-CHO for 1 h and exposed to BetA for 48 h, then lysed in sample buffer for detection. Western blot analysis of t-Akt and p-Akt levels was shown. Right , Western blot analysis of the effect of DEVD-CHO (100 μM) in PP2A cleavage in IM-9 cells. (B) Detection of the binding of Akt or DAPK with PP2A in IM-9 cells. Cells were incubated with or without BetA for 48 h. In some groups of cells, 0.2 μM of OA or 100 μM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with for immunoprecipitation with anti-Akt or anti-DAPK antibody followed by immunoblot assay with anti- PP2A/C and anti-Akt or anti-DAPK antibodies. (C) Detection of the binding of Akt or DAPK with PP2A in IM-9/Bcl-2 cells. Cells were incubated with or without BetA for 48 h. In some groups of cells, 0.2 μM of OA or 100 μM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with for immunoprecipitation with anti-Akt or anti-DAPK antibody followed by immunoblot assay with anti- PP2A/C and anti-Akt or anti-DAPK antibodies. Representative results of three experiments with consistent results are shown.
Techniques Used: Incubation, Western Blot, Binding Assay, Immunoprecipitation
![BetA induces autophagic cell death in IM-9/Bcl-2 not in IM-9 cells (A) immunoblot analysis of LC3-I and LC3-II levels in cells. Cells were treated as described in Figure 1 , and then treated cells were collected for detecting LC3-I and LC3-II content by Western blotting, with β-Actin serving as a loading control. (B) Cells were treated as described in A . IM-9/Bcl-2/GFP-LC33 cell lines were treated with BetA for 12 h, fixed, and then visualized by fluorescent microscopy, Bars, 10 μM. (C) Autophagosome inhibitor 3-MA attenuated the effect of BetA on autophagy in IM-9/Bcl-2 cells. Cells were treated with BetA for 48 h in the absence or presence of the inhibitor of class III PI3 kinases 3-MA (5 mM), lysed and subjected to western blotting with anti-LC3 or p62 antibodies to monitor autophagy. EBSS, (starvation medium) is as a control, which is a classical stimulus used to induce the build-up of autophagosomes and autophagic flux. (D) BetA promotes long-lived protein degradation in cells cultured in full medium. IM-9 and IM-9/Bcl-2 cells were first transfected with Ctrl or ATG5 siRNA-1 for 24 h, and then radiolabeled for 24 h with 0.05 mCi/ml of L-[U- 14 C]valine. At the end of the labeling period, the cells were rinsed three times with phosphate-buffered saline. The cells were then incubated in full medium in the presence or in the absence of 10 μg/ml BetA or EBSS with 10 mM valine for 12 h. The data are presented as the means ± S.D. from three independent experiments (*, P](https://storage.googleapis.com/bioz_article_images/PMC5655238/oncotarget-08-80770-g002.jpg "BetA induces autophagic cell death in IM-9/Bcl-2 not in ...")
Figure Legend Snippet: BetA induces autophagic cell death in IM-9/Bcl-2 not in IM-9 cells (A) immunoblot analysis of LC3-I and LC3-II levels in cells. Cells were treated as described in Figure 1 , and then treated cells were collected for detecting LC3-I and LC3-II content by Western blotting, with β-Actin serving as a loading control. (B) Cells were treated as described in A . IM-9/Bcl-2/GFP-LC33 cell lines were treated with BetA for 12 h, fixed, and then visualized by fluorescent microscopy, Bars, 10 μM. (C) Autophagosome inhibitor 3-MA attenuated the effect of BetA on autophagy in IM-9/Bcl-2 cells. Cells were treated with BetA for 48 h in the absence or presence of the inhibitor of class III PI3 kinases 3-MA (5 mM), lysed and subjected to western blotting with anti-LC3 or p62 antibodies to monitor autophagy. EBSS, (starvation medium) is as a control, which is a classical stimulus used to induce the build-up of autophagosomes and autophagic flux. (D) BetA promotes long-lived protein degradation in cells cultured in full medium. IM-9 and IM-9/Bcl-2 cells were first transfected with Ctrl or ATG5 siRNA-1 for 24 h, and then radiolabeled for 24 h with 0.05 mCi/ml of L-[U- 14 C]valine. At the end of the labeling period, the cells were rinsed three times with phosphate-buffered saline. The cells were then incubated in full medium in the presence or in the absence of 10 μg/ml BetA or EBSS with 10 mM valine for 12 h. The data are presented as the means ± S.D. from three independent experiments (*, P
Techniques Used: Western Blot, Microscopy, Cell Culture, Transfection, Labeling, Incubation
Figure Legend Snippet: Beclin-1 is required for autophagic cell death in IM-9/Bcl-2 cells (A) Detection of Beclin-1/Bcl-2 complex and Beclin-1/Vps 34 complex association in IM-9/Bcl-2 cells, respectively. Top row , Cells were cultured for 24 h, either in complete medium (control) or in medium supplemented with 10 μg/ml BetA. EBSS treatment (12 h) was used as an autophagy control. Cells were lysed and were subjected to immunoprecipitation with anti-Beclin 1 antibody and protein G-Sepharose. The immunoprecipitates were subjected to immunoblotting using anti-Bcl-2, Vps34 or anti-Beclin 1 antibodies. Bottom , lysates were immunoblotted with the antibodies indicated. The Western blots are representative of three independent experiments. (B) IM-9/Bcl-2 cells were transfected with Beclin-1 or Ctrl siRNA for 48 h, and then treated with BetA for different periods of time. Immunoblot analysis of LC3-I and LC3-II levels, p62 and Beclin-1 expression. Arrowhead referred to the upshifted Beclin-1. β-Actin immunoblotting was used as a loading control. (C) IM-9/Bcl-2 cells were treated as described in B , and then treated cells were collected for MDC staining by flow cytometry. Representative results of three experiments with consistent results are shown. (D) IM-9 and IM-9/Bcl-2 cells were transfected with Beclin-1 or Ctrl siRNA for 48 h, and then treated with BetA for different periods of time. Cell viability was determined as described in Materials and methods. Graphs showing results of quantitative analyses ( n =3, mean ± S.D. **, P
Techniques Used: Cell Culture, Immunoprecipitation, Western Blot, Transfection, Expressing, Staining, Flow Cytometry, Cytometry
Figure Legend Snippet: DAPK-mediated Beclin-1 phosphorylation contributes to BetA-induced autophagic cell death (A) IM-9 and IM-9/ Bcl-2 cells were treated with BetA for different periods of time. Treated cells were lysed for detecting DAPK phosphorylation (p-DAPK), total DAPK (t-DAPK), Beclin-1 phosphorylation (p-Beclin-1) and total Beclin-1 (t-Beclin-1) by Western blotting, with β-Actin serving as a loading control. Arrowhead referred to the upshifted Beclin-1. (B) IM-9/ Bcl-2 cells were transfected with Ctrl vector or HA-Beclin-1 T119A for 48 h, and then cells were lysed for detecting LC3-I and LC3-II levels by Western blotting, with β-Actin serving as a loading control. (C) IM-9/ Bcl-2 cells were treated with BetA for 48 h, and then treated cells were collected for MDC staining by flow cytometry. Representative results of three experiments with consistent results are shown. (D) IM-9/ Bcl-2 cells were treated as described in B , and cell viability was determined as described in Materials and methods. Graphs showing results of quantitative analyses ( n =3, mean ± S.D. **, P
Techniques Used: Western Blot, Transfection, Plasmid Preparation, Hemagglutination Assay, Staining, Flow Cytometry, Cytometry
Figure Legend Snippet: PP2A is required for BetA-induced apoptosis or autophagic cell death (A) Cells were treated by BetA for different periods of time, and then lysed for Western blotting with β-Actin serving as a loading control. Treated cells were lysed with sample buffer and subjected to immunoblot assay with an A subunit-specific anti-PP2A/A antibody. CF is referred to cleaved PP2A/A. The membrane was then stripped and reprobed with a C subunit-specific anti-PP2A/C antibody. β-Actin was used as a loading control. (B) Different cells were pre-incubated with indicated concentrations of OA for 1 h and exposed to BetA for 48 h, then lysed for detection. (C) Cells were transiently transfected with PP2A/C or Ctrl siRNA for 48 h, and then treated by BetA for Western blot analysis. (D) Cells were treated with OA or transfected with PP2A/C siRNA, and then treated with BetA for 48 h. Treated cells were collected for detection of cell viability. Graphs showing results of quantitative analyses ( n =3, mean ± S.D. *, P
Techniques Used: Western Blot, Incubation, Transfection
Figure Legend Snippet: BetA induces apoptosis-independent cell death in IM-9/Bcl-2 and apoptosis in IM-9 cancer cells (A) Analysis of cell viability treated with BetA. IM-9 and IM-9/Bcl-2 cells were treated with different concentrations of BetA for 48 h. Cell viability was determined as described in Materials and methods. A minimum of 100 cells per sample for triplicate samples was counted per condition per experiment. Graphs showing results of quantitative analyses ( n =3, mean ± S.D. **, P
Techniques Used:
2) Product Images from "Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death"
Article Title: Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death
Journal: Cell Death & Disease
doi: 10.1038/cddis.2017.130
Figure Legend Snippet: Fluphenazine induces HIF overactivation in conditions of high HIF background levels. ( a ) HIF activity reporter cells HCT116-HRE-Luc were treated either with DMSO control or 10 μ M Fluphenazine and incubated for 16 h in normoxia, hypoxia or in normoxia with the HIF-PH inhibitor DFO. After incubation, cells were lysed and luciferase activity was measured. Bars show mean with S.D. ( n =3). ( b ) HCT116 cells were treated for 24 h with or without DFO (1 mM) and additionally with either DMSO control or Fluphenazine (5 μ M). Gene expression analysis was performed for three HIF1 target genes (SLC2A3, VEGFA and BNIP3) by real-time quantitative PCR (RT-qPCR). hRP-L32 was used as reference gene and relative expression level were normalized to the untreated control (no DFO, DMSO). Bars show mean with S.D. ( n =3). ( c ) HCT116-HRE-Luc cells were treated with either Ethanol control or 100 μ M SM (18:1/16) and incubated for 24 h either in normoxia or hypoxia. After incubation, cells were lysed and luciferase activity was measured ( n =3). ( d ) RT-qPCR analysis of HIF-1-a mRNA level of HCT116 Spheroids treated for 24 h in normoxia or hypoxia with either DMSO control or 5 μ M Fluphenazine. hRP-L32 was used as reference gene and the relative expression level were normalized with the untreated control (normoxia DMSO control). Bars show mean with S.D. ( n =3). ( e ) Western blotting analysis of HIF-1-a protein expression in HCT116 spheroids or HCT116 cells grown in 2D that were treated for 24 h with either DMSO control or 5 μ M Fluphenazine under normoxia, hypoxia or normoxia+1 mM DFO. Representative data of multiple experiments are shown ( n =3). Intensity values were normalized to loading control and DMSO-treated controls under hypoxia (upper row) or DMSO controls co-incubated with DFO (lower row). Beta-actin was used as an internal control (not shown). * = P -value between 0.01 and 0.05, ** = P -value between 0.001 and 0.01, *** = P -value between 0.0001 and 0.001, **** = P -value smaller 0.0001
Techniques Used: Activity Assay, Incubation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot
Figure Legend Snippet: HCT116 tumor spheroids incubated in hypoxia mimic hypoxic tumor regions distal from blood vessels in HCT116 xenografts. ( a ) IHC staining of HCT116 colon cancer xenograft tumor sections. HCT116 tumor cryosections were stained for the exogenous hypoxia marker pimonidazole (Hypoxyprobe, green), CD31 as marker for blood vessel (red) and the nuclear marker Hoechst (blue). Arrows indicate blood vessels. N=necrotic region. Dashed line showing region for measuring intensity profile in panel ( b ). Scale bar 100 μ m. ( b ) Line scan (dashed line in panel ( a )) through HCT116 tumor section showing intensity profile of pimonidazole and CD31 staining. ( c ) Cryosections of HCT116 spheroids. Spheroids were treated for 3 days in normoxia or hypoxia with or without the complex III inhibitor Antimycin (200 nM). Nuclei were stained by Hoechst (blue) and hypoxic areas with anti-pimonidazole (green). Scale bar 100 μ m. ( d ) Intensity profile pimonidazole staining (average of multiple spheroids, n =5–10) from spheroid border to spheroid core region of HCT116 spheroids. ( e ) Western blotting analysis of HCT116 spheroids incubated for 24 h in normoxia or hypoxia. Beta-actin was used as an internal control. Representative data of multiple experiments shown ( n =3). ( f ) Real-time quantitative PCR gene expression analysis of HIF target genes in HCT116 spheroids incubated for 24 h in normoxia or hypoxia. Ct values of each sample were normalized with the internal control RPL32 and normalized to the normoxia sample. Bars show mean with S.D. ( n =3). **** = P -value smaller 0.0001
Techniques Used: Incubation, Immunohistochemistry, Staining, Marker, Western Blot, Real-time Polymerase Chain Reaction, Expressing
3) Product Images from "Global transcriptional silencing and developmental competence in the oocyte mediated by the mRNA decay activator ZFP36L2"
Article Title: Global transcriptional silencing and developmental competence in the oocyte mediated by the mRNA decay activator ZFP36L2
Journal: Developmental cell
doi: 10.1016/j.devcel.2018.01.006
Figure Legend Snippet: ZFP36L2 expression in oocytes is required for female fertility and oocyte developmental competence (A) Relative level of Zfp36l2 mRNA by qPCR analysis in cKO and Fl/Fl GV oocytes, normalized to Gapdh . Error bars are SEM (unpaired T-test; ***, p < 0.001). (B) Western blot comparing ZFP36L2 in cKO and Fl/Fl GV oocytes. Beta-actin is the internal loading control. (C) Box plot showing the distribution of the number of pups/litter for cKO and controls over a six-month period. ##, no pups. (D) Hemotoxylin and Eosin stained sections of ovaries from unstimulated adult Fl/Fl and cKO females. Pr, primordial; 1°, primary; 2°, secondary; Antral, antral; CL, corpus luteum. Scale bar, 50 um. (E) Number of follicles at each stage of development in whole ovaries of mature unstimulated Fl/Fl and cKO mice. Bars represent mean and SEM at each stage of development (unpaired T-test. *, p < 0.05). (F) Number of GV, MI and MII oocytes isolated from cKO and Fl/Fl females following superovulation in vivo as a percent of total oocytes (unpaired T-test; *, p < 0.05). Insets, MII oocyte enlarged to show the abnormally large polar bodies observed in many cKO oocytes. Scale bar, 100 μm. (G) Number of zygotes isolated from cKO and Fl/Fl females following superovulation and mating (unpaired T-test; ***, p < 0.001. ##, no zygotes. Arrowheads in top left panel indicate pronuclei identified only in cKOs. Scale bar, 100 μm. See also .
Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Mouse Assay, Isolation, In Vivo
4) Product Images from "Necroptosis in primate luteolysis: a role for ceramide"
Article Title: Necroptosis in primate luteolysis: a role for ceramide
Journal: Cell Death Discovery
doi: 10.1038/s41420-019-0149-7
Figure Legend Snippet: Proteomic analysis and verification of involvement of the endogenous ceramide system by FB1 blocking experiments. Levels of ceramide salvage pathway proteins increased during culture in human IVF-derived GCs. a Illustration of enzymatic reactions of ceramide within the salvage pathway in the lysosome, and ceramide synthesis in the endoplasmatic reticulum. Three different molecules get processed enzymatically to ceramide; Sphingomyelin by the acid sphingomyelinase, D-galactocerebroside by galactosylceramidase and glucocerebroside by acid beta-glucosidase. Ceramide then gets turned over by acid ceramidase to sphingosine, which can be processed back to ceramide in the endoplasmatic reticulum 61 . Proteins in grey were not significantly altered in abundance. Quantified proteins were colored with the shown gradient according to their log 2 fold change between day 5 and day 2. b Volcano plot comparing day 5 with day 2 of human GC culture. Red marked proteins differed significantly in abundance after multiple testing correction. Table 2 shows the corresponding log 2 fold changes as well as the p values. For each time point 5 GC samples were measured. c GCs, cultured on glass coverslips were fixed on day 2 or day 5 of culture. Cells were also stimulated with fumonisin B1 (FB1, 0.5 µM, 72 h) before fixation. Only merged pictures are shown, separated channels are available in Supplementary Fig. 4a . Immunocytochemical staining using anti-ceramide and anti-golgin97 antibodies revealed different intensities and cellular localization, depending on culture time and stimulation. Controls using rabbit IgG and mouse IgM are shown in inset of the first picture. Two experiments were performed and representative pictures are shown. Scale bars indicate 25 µm. d Cell numbers of GCs that were treated with FB1 (0.5 µM, n = 11) for 72 h are depicted in the bar diagram. On the right side, confluency data of FB1-stimulated stimulated GCs (0.5 µM, n = 6, a) are depicted, which were collected every 20 min and normalized to the corresponding solvent control. Paired Student’s t test was conducted to evaluate statistical significance (* p
Techniques Used: Blocking Assay, Derivative Assay, Gas Chromatography, Cell Culture, Staining
5) Product Images from "Self-adjusting synthetic gene circuit for correcting insulin resistance"
Article Title: Self-adjusting synthetic gene circuit for correcting insulin resistance
Journal: Nature biomedical engineering
doi: 10.1038/s41551-016-0005
Figure Legend Snippet: Synthetic insulin-sensitizing designer circuit for the treatment of insulin resistance. (a) Functional and therapeutic features. Insulin resistance is characterized by the insensitivity of liver, muscle and adipocytes to blood insulin levels, which prevents cellular glucose uptake and leads to hyperinsulinaemia. The designer circuit quantifies blood insulin levels, detects hyperinsulinaemia and triggers dose-dependent adiponectin expression, which restores insulin sensitivity and attenuates the insulin-resistance syndrome. (b) Synthetic insulin-sensitizing designer cascade. The binding of insulin to the tyrosine kinase family human insulin receptor (IR) triggers autophosphorylation on multiple tyrosine residues in the IR, leading to the phosphorylation of cellular proteins such as insulin receptor substrate 1 (IRS-1). Upon tyrosine phosphorylation, these proteins interact with signaling molecules through their SH 2 domains, resulting in subequent activation of Ras (a GTPase) and mitogen-activated protein kinase (MAPK). The activated MAPK enters the nucleus and phosphorylates the synthetic hybrid transcription factor, TetR-ELK1, consisting of the doxycycline-responsive DNA-binding Tet repressor (TetR) fused to the regulated activation domain of the transcription factor, ELK1, which is driven by the constitutive human cytomegalovirus immediate early promoter (P hCMV ). In the basal state, TetR-ELK1, is able to bind to P hCMV*-1 , a chimeric promoter containing a TetR-specific heptameric operator module (tetO 7 ) linked to a minimal version of P hCMV (P hCMVmin ), but the ELK1 domain remains inactive. Only the phosphorylation of TetR-ELK1’s ELK1 domain by upstream MAPK activity during cell signaling leads to P hCMV*-1 -driven expression of the desired transgene. The addition of doxycycline disrupts the interaction between TetR-ELK1’s TetR domain and the tetO 7 operator of P hCMV*-1 , preventing the hybrid transcription factor from mediating the transgene expression, regardless of its phosphorylation state. When set to produce a therapeutic protein such as adiponectin that is secreted into the bloodstream the designer cascade turns into a closed-loop prosthetic network that increases the sensitivity of liver, adipose and muscle tissues to insulin which attenuates insulin release by pancreatic beta cells and reverses the insulin resistance syndrome.
Techniques Used: Functional Assay, Expressing, Binding Assay, Activation Assay, Activity Assay
6) Product Images from "Autophagy flux induced by ginsenoside-Rg3 attenuates human prion protein-mediated neurotoxicity and mitochondrial dysfunction"
Article Title: Autophagy flux induced by ginsenoside-Rg3 attenuates human prion protein-mediated neurotoxicity and mitochondrial dysfunction
Journal: Oncotarget
doi: 10.18632/oncotarget.13730
Figure Legend Snippet: Rg3 protects neuronal cells via autophagic pathway ( A ) SK-N-SH neuroblastoma cells were pretreated with 25 μM of Rg3 in the presence of autophagy inhibitors (wortmannin or 3MA) for 6 h and then exposed to 100 μM of PrP (106-126) for 12 h. Cell viability was determined using Annexin V assay. ( B ) Bar graph indicating the averages of annexin V-negative cells. ( C ) SK-N-SH cells were mixed with a titration (30MOI) of BacMam GFP-LC3B virus for 18 h. They were then treated with Rg3 and autophagy inhibitors for 6h. Negative control reagent and positive control reagent (CQ) were included at the same time. ( D ) Western blot analysis for LC3B production and p62/SQSTM1 expression in SK-N-SH cells. Beta-actin was used as a loading control. ( E ) Bar graph indicating the averages of LC3B-II expression levels. ( F ) SK-N-SH cells were incubated with Rg3 at 25 μM and wortmannin for 6 h followed by TEM. Arrowheads indicate autophagosomes. * p
Techniques Used: Annexin V Assay, Titration, Negative Control, Positive Control, Western Blot, Expressing, Incubation, Transmission Electron Microscopy
Figure Legend Snippet: ATG5 knockdown decreases Rg3-mediated neuroprotective effect ( A ) ATG5 small interfering RNA (siATG5) or negative control siRNA (NC)-transfected SK-N-SH cells were incubated with PrP (106-126) for 12 h in the presence of Rg3. Cell viability was determined with Annexin V assay. ( B ) Bar graph indicating the averages of annexin V-negative cells. ( C ) Western blot analysis for ATG5 and LC3B production in SK-N-SH cells. Beta-actin was used as a loading control. ( D ) Bar graph indicating the averages of LC3B-II expression levels. ** p
Techniques Used: Small Interfering RNA, Negative Control, Transfection, Incubation, Annexin V Assay, Western Blot, Expressing
7) Product Images from "E-cadherin can limit the transforming properties of activating β-catenin mutations"
Article Title: E-cadherin can limit the transforming properties of activating β-catenin mutations
Journal: The EMBO Journal
doi: 10.15252/embj.201591739
Figure Legend Snippet: Apc loss, GSK3 loss and homozygous mutation of β-catenin are sufficient to induce a rapid crypt-progenitor phenotype, but not a single β-catenin mutation Wild-type mice have small defined crypts in the small intestine with little nuclear β-catenin at the bottom of the crypt. The small intestine of AhCre ER APC fl/fl , AhCre ER Catnb lox(ex3)/lox(ex3) (day 5) or AhCre ER Gsk3alpha fl/fl beta fl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclear β-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm. A heterozygous activation of β-catenin ( AhCre ER Catnb lox(ex3)/ + ) shows no increase in crypt size or nuclear β-catenin at days 5–10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm. Culture of small intestinal crypts of WT and VilCre ER Apc fl/fl (or AhCre ER Apc fl/fl ) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc -deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm. At day 5 post-induction, only crypts from AhCre ER Catnb lox(ex3)/lox(ex3) but not AhCre ER Catnb lox(ex3)/ + survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCre ER Catnb lox(ex3)/ + compared to spheres from Catnb lox(ex3)/lox(ex3) crypts in the first week of culture. Black scale bar, 50 μm. Quantification of organoids/spheres at day 10 post-induction ( N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).
Techniques Used: Mutagenesis, Mouse Assay, Activation Assay
Figure Legend Snippet: Only deletion of both Gsk3alpha and Gsk3beta leads to crypt-progenitor-cell phenotype Small Intestine of mice at day 6 after induction. Loss of Gsk3alpha and Gsk3beta ( AhCre ER Gsk3alpha fl/fl Gsk3beta fl/fl ) leads to accumulation of nuclear β-catenin and upregulation of cMyc (arrows). The crypt-progenitor cell phenotype (red bar) is characterised by increased proliferation (BrdU) and perturbed differentiation/localisation of goblet and Paneth cells (Alcian Blue, Lysozyme respectively, arrows). Scale bar, 100 μm. Table shows cohort of AhCre Gsk3alpha Gsk3beta mice aged until signs of intestinal tumour burden, genotypes as indicated. Note, mice homozygous for Gsk3beta deletion, or with only one copy of GSK3alpha and GSK3beta , did not develop intestinal tumours. An adenoma from an aged mouse deficient for 3 alleles of Gsk3alpha and Gsk3beta (AhCre GSK3alpha fl/fl beta fl/ + ) showing that it developed after loss of the remaining GKS3beta allele. Scale bar, 100 μm.
Techniques Used: Mouse Assay
8) Product Images from "Protective effect of clusterin on rod photoreceptor in rat model of retinitis pigmentosa"
Article Title: Protective effect of clusterin on rod photoreceptor in rat model of retinitis pigmentosa
Journal: PLoS ONE
doi: 10.1371/journal.pone.0182389
Figure Legend Snippet: Up-regulation of STAT3 by clusterin in RP retina. The phosphorylated STAT3 immunoreactive protein was examined by immunoblot analysis in saline- and clusterin-treated RP retinas (A). Retinas were collected at 5 min, 1 hour, 6 hours, and 24 hours after injection at P15. Expression of pSTAT3 was up-regulated at 5 min. and peaked at 6hrs after injection of clusterin (+). In contrast, saline-treated retinas (-) showed slight activation of STAT3 throughout the time points after injection. Densitometry analysis of pSTAT3 was shown by measuring the intensity relative to control β-actin (B). Beta-actin and STAT3 served as the loading control. Confocal micrographs of immunoreactivity of pSTAT3 (red) and GS (green) in vertical sections of P15 RP retina, which were collected 6 hours after saline and clusterin injection (C). In saline-treated RP retina, no pSTAT3 immunoreactivity was observed. In clusterin-treated RP retina, pSTAT3 immunoreactivity was detected in the processes in the ONL and INL. Double-labeling experiments showed that processes of Müller cells and their cell bodies were co-localized with pSTAT3 immunoreactivity (arrows). In addition, some processes of Müller cells in the ONL were also labeled with pSTAT3 (arrowheads). Bars mean ± SEM, *P = 0.0224, **P = 0.0031, *** P
Techniques Used: Injection, Expressing, Activation Assay, Labeling
Figure Legend Snippet: Induction of pAkt by clusterin in RP retina. Immunoblot analysis of phosphorylated Akt (60kDa) was examined in saline- and clusterin-treated RP retinas (A). Retinas were collected at 5 min, 1 hour, 6 hours, and 24 hours after injection at P15. Activation of Akt was detected as early as 5 min after clusterin injection. The saline-treated retinas (-) showed lower activation of Akt at different time points, compared to the clusterin-treated retinas (+). Densitometry analysis of immunoblots for phosphorylation of Akt by clusterin was generated in histogram (B). Beta-actin served as the loading control to gain relative pAkt activation value. Data represents mean ± SEM, *** P
Techniques Used: Injection, Activation Assay, Western Blot, Generated
9) Product Images from "Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis"
Article Title: Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis
Journal: Oncogene
doi: 10.1038/onc.2015.302
Figure Legend Snippet: Mechanism of PTGS2 induction downstream of norepinephrine (NE) A, Skov3-ip1 (left) and HeyA8 (right) cells were treated with NE, isoproterenol (a broad ADRB agonist), and specific beta-agonists (ADRB1: dobutamine; ADRB2: terbutaline; ADRB3: CL316423) and assessed for expression of PTGS2 and PTGES. B, Skov3-ip1 (left) and HeyA8 (right) cells were treated with propranolol (broad ADRB antagonist) and butoxamine (ADRB2-specific antagonist) prior to treatment with NE. Isoproterenol and terbutaline were included as positive controls. C, Enzyme-linked immunosorbent assay results showing PGE2 levels in conditioned media from Skov3-ip1 (left) and HeyA8 (right) cells treated with NE, isoproterenol, terbutaline, propranolol plus NE, and butoxamine plus NE. D, Chromatin was obtained from Skov3-ip1 cells treated with NE and incubated with specific Nf-kB antibodies. Quantitative reverse-transcription polymerase chain reaction was then used to assess fold enrichment of Nf-kB on the promoters of PTGS2 and PTGES, normalized to non-treated (NT) cells. E, Nuclear proteins were isolated from Skov3-ip1 cells treated with NE and levels of p65 and p50 in the nucleus were compared with levels in NT cells, assessed as fold changes. In all experiments, n = 3 per group and data are presented as mean ± standard error of the mean. Statistical significance was obtained using the Student t test: *p
Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Isolation
10) Product Images from "Identification and characterization of transforming growth factor beta‐induced in circulating tumor cell subline from pancreatic cancer cell line. Identification and characterization of transforming growth factor beta‐induced in circulating tumor cell subline from pancreatic cancer cell line"
Article Title: Identification and characterization of transforming growth factor beta‐induced in circulating tumor cell subline from pancreatic cancer cell line. Identification and characterization of transforming growth factor beta‐induced in circulating tumor cell subline from pancreatic cancer cell line
Journal: Cancer Science
doi: 10.1111/cas.13783
Figure Legend Snippet: Suppression of transforming growth factor beta‐induced (TGFBI) decreased the migration and invasion abilities of Panc‐1‐circulating tumor cells (CTC). A,B, Knockdown of TGFBI expression in Panc‐1‐CTC cells by TGFBI‐specific siRNA as confirmed by qRT‐PCR (A) and western blotting (B) with samples 3 d after transfection with siRNA. C, Number of viable cells 24‐72 h after transfection of TGFBI‐specific siRNA or a negative control siRNA in Panc‐1‐CTC cells was determined by WST‐8 assay at the indicated times. Results of the relative growth ratio are shown as the mean ± SD for three separate experiments, with each carried out in triplicate. Differences were analyzed by one‐way ANOVA. D,E, Numbers of migratory (D) and invasive cells (E) through the Boyden chamber at 24 h after replating (1.5 × 10 4 cells per well) 3 d after siRNA transfection were counted using Diff‐Quick Stain. Data are presented as mean ± SD of samples (n = 3). Differences were analyzed by the Mann‐Whitney U test. F, TGFBI expression in 25 pancreatic cancer cell lines was assessed by qRT‐PCR in comparison with healthy pancreatic tissue. Data are presented as mean ± SD of samples (n = 3)
Techniques Used: Migration, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Diff-Quik, Staining, MANN-WHITNEY
Figure Legend Snippet: Overexpression of transforming growth factor beta‐induced ( TGFBI ) increased the migration and invasion abilities of Panc‐1‐parent (Panc‐1‐P) cells. A,B, Overexpression of TGFBI in Panc‐1‐P cells stably transfected with pCMV3Tag3A‐TGFBI expression vector as confirmed by qRT‐PCR (A) and western blotting (B). Protein expression of exogenous TGFBI was confirmed with anti‐FLAG antibody. C, Number of viable cells 24‐96 h in Panc‐1‐P cells transduced with empty vector and TGFBI was determined by WST‐8 assay at the indicated times. Results of the relative growth ratios are shown with mean ± SD for three separate experiments, each carried out in triplicate. Differences were analyzed by one‐way ANOVA. D, Number of tumorsphere formations was counted if the length was over 200 μm. Data are presented as mean ± SD of samples (n = 4). Differences were analyzed by t test. E,F, Number of migratory (E) and invasive cells (F) through the Boyden chamber at 24 h after replating was counted using Diff‐Quick Stain. Data are presented as mean ± SD of samples (n = 3). Differences were analyzed by the Mann‐Whitney U test
Techniques Used: Over Expression, Migration, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Transduction, Diff-Quik, Staining, MANN-WHITNEY
Figure Legend Snippet: Knockdown of transforming growth factor beta‐induced ( TGFBI ) expression decreased the migration and invasion abilities of CAPAN‐1 and CFPAC‐1 cells. A,B, Knockdown of TGFBI expression in CAPAN‐1 and CFPAC‐1 cells by TGFBI‐specific siRNA as confirmed by qRT‐PCR (A) and western blotting (B) with samples 3 d after siRNA transfection. C, Number of viable cells 24‐72 h after transfection of TGFBI‐specific siRNA or a negative control siRNA in CAPAN‐1 and CFPAC‐1 cells was determined by WST‐8 assay at the indicated times. Results of the relative growth ratios are shown with mean ± SD for three separate experiments, with each carried out in triplicate. Differences were analyzed by one‐way ANOVA. D,E, Numbers of migratory (D) and invasive cells (E) through the Boyden chamber at 24 h after replating (1.5 × 10 4 cells per well) at 3 d after siRNA transfection were counted using Diff‐Quick Stain. Data are presented as mean ± SD of samples (n = 3). Differences were analyzed by the Mann‐Whitney U test
Techniques Used: Expressing, Migration, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Diff-Quik, Staining, MANN-WHITNEY
Figure Legend Snippet: Expression of transforming growth factor beta‐induced (TGFBI) was associated with poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). A, Representative images of immunohistochemical staining of TGFBI protein in PDAC tissues. Bars, 100 μm. B,C, Kaplan‐Meier curves for the overall survival (B) and disease‐free survival (C) of 75 patients with primary PDAC. High TGFBI immunoreactivity in tumor tissues was significantly associated with poor overall and disease‐free survival ( P
Techniques Used: Expressing, Immunohistochemistry, Staining
![Expression of transforming growth factor beta‐induced (TGFBI) in Panc‐1‐circulating tumor cells (CTC) was increased in both in vitro and in vivo samples. A, Subcutaneous tumor size of Panc‐1‐parent (Panc‐1‐P) and Panc‐1‐CTC cells was measured every week after inoculation using the formula tumor size (mm 3 ) = [(length) × (width) 2 ]/2. Data are presented as mean ± SD of samples (n = 4). B, Mice were killed on day 28 after inoculation, and the weights of the s.c. tumors were measured. Data are presented as a box plot ± SD of the samples (n = 4). C, Results of the gene expression array with in vitro and in vivo samples of Panc‐1‐P and Panc‐1‐CTC cells. D,E, In vitro TGFBI expression was assessed by qRT‐PCR (D) and western blotting (E). F,G, In vivo TGFBI expression was assessed by qRT‐PCR (F) and immunohistochemical staining with each s.c. tumor derived from Panc‐1‐P or Panc‐1‐CTC cells (G). G, Representative images of immunohistochemical staining of TGFBI protein in s.c. tumorous tissues from mice. Bars, 100 μm](https://storage.googleapis.com/bioz_article_images/PMC6215881/CAS-109-3623-g002.jpg "Expression of transforming growth factor beta‐induced (TGFBI) in Panc‐1‐circulating tumor cells (CTC) was increased ...")
Figure Legend Snippet: Expression of transforming growth factor beta‐induced (TGFBI) in Panc‐1‐circulating tumor cells (CTC) was increased in both in vitro and in vivo samples. A, Subcutaneous tumor size of Panc‐1‐parent (Panc‐1‐P) and Panc‐1‐CTC cells was measured every week after inoculation using the formula tumor size (mm 3 ) = [(length) × (width) 2 ]/2. Data are presented as mean ± SD of samples (n = 4). B, Mice were killed on day 28 after inoculation, and the weights of the s.c. tumors were measured. Data are presented as a box plot ± SD of the samples (n = 4). C, Results of the gene expression array with in vitro and in vivo samples of Panc‐1‐P and Panc‐1‐CTC cells. D,E, In vitro TGFBI expression was assessed by qRT‐PCR (D) and western blotting (E). F,G, In vivo TGFBI expression was assessed by qRT‐PCR (F) and immunohistochemical staining with each s.c. tumor derived from Panc‐1‐P or Panc‐1‐CTC cells (G). G, Representative images of immunohistochemical staining of TGFBI protein in s.c. tumorous tissues from mice. Bars, 100 μm
Techniques Used: Expressing, In Vitro, In Vivo, Mouse Assay, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Derivative Assay
11) Product Images from "Tra2-Mediated Recognition of HIV-1 5? Splice Site D3 as a Key Factor in the Processing of vpr mRNA"
Article Title: Tra2-Mediated Recognition of HIV-1 5? Splice Site D3 as a Key Factor in the Processing of vpr mRNA
Journal:
doi: 10.1128/JVI.02756-12
Figure Legend Snippet: HIV-1 exon 3 splicing is under the combined control of ESSV and ESEvpr . Splice site recognition at HIV-1 exon 3 is regulated by ESSV and a novel exonic splicing enhancer (ESEvpr ) embedded in the region upstream of 5′ss D3. ESSV is associated with hnRNP A/B proteins, which may multimerize along the 5′ end of exon 3, occluding 3′ss A2. Tra2-alpha and Tra2-beta bind to the ESEvpr sequence, potentially enhancing recruitment of the U1 snRNP to 5′ss D3, which in turn may promote interactions across the upstream exon and activation of 3′ss A2. U1, U1 snRNP.
Techniques Used: Sequencing, Activation Assay
12) Product Images from "Assessment of the role of translationally controlled tumor protein 1 (TPT1/TCTP) in breast cancer susceptibility and ATM signaling"
Article Title: Assessment of the role of translationally controlled tumor protein 1 (TPT1/TCTP) in breast cancer susceptibility and ATM signaling
Journal: Clinical and Translational Radiation Oncology
doi: 10.1016/j.ctro.2019.01.006
Figure Legend Snippet: Identification, screening and prediction of functional impact of c.452A > G (p.Y151C). A: Identification of mutation c.452A > G (p.Y151C) by Sanger sequencing in a heterozygous breast cancer patient (right, arrow) compared with the wild-type sequence (left). B: High Resolution Melting analysis showing melting curves of the two variants c.452A > G (p.Y151C), c.492G > A, and wild-type samples. C: 3D-model of TPT1 by Polyphen-2 showing altered amino acid as part of a beta sheet structure. D,E: Prediction results by SIFT-analysis (D) and PROVEAN-analysis (E) showing damaging impact of c.452A > G (p.Y151C) F: comparison of prediction tool performance.
Techniques Used: Functional Assay, Mutagenesis, Sequencing
13) Product Images from "Heparan sulfate proteoglycans present PCSK9 to the LDL receptor"
Article Title: Heparan sulfate proteoglycans present PCSK9 to the LDL receptor
Journal: Nature Communications
doi: 10.1038/s41467-017-00568-7
Figure Legend Snippet: Enzymatic removal of liver heparan sulfate releases PCSK9 and ablates its activity. a , b Infusion of heparinase I prior to the injection of PCSK9 (10 µg) completely inhibits PCSK9-induced degradation of LDLR. Western blot of representative samples is shown in a and quantification of LDLR in b (control n = 7, PCSK9 n = 6, heparinase n = 5, heparinase/PCSK9 n = 5). Heparinase treatment unmasks the antigenicity of the major liver HSPG syndecan-1 ( middle panel ). Beta-actin is used as loading control ( lower panel ). c Heparinase I treatment leads to an increase in plasma PCSK9 as measured by ELISA 15 min after injection (control n = 6, heparinase n = 6). d Western blot (representative samples) of liver syndecan-1 is used as control of heparinase injection. Beta-actin is shown as loading control. e – h Transgenic mice with constitutive expression of human heparanase ( Hpa -tg) ( n = 7) have increased plasma PCSK9 ( e ), increased liver LDLR ( f , g ), and reduced plasma cholesterol ( h ) compared to control WT mice ( n = 6). Bar graphs show mean with s.e.m. error bars. Statistical significance was evaluated using a two-tailed Student’s t -test. Supplementary Fig. 11 shows uncropped gel images
Techniques Used: Activity Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Mouse Assay, Expressing, Two Tailed Test
14) Product Images from "A GCase Chaperone Improves Motor Function in a Mouse Model of Synucleinopathy"
Article Title: A GCase Chaperone Improves Motor Function in a Mouse Model of Synucleinopathy
Journal:
doi: 10.1007/s13311-014-0294-x
Figure Legend Snippet: GCase activity and AT2101 effects in human wild-type (WT) α-synuclein (Thy1-aSyn) mice. a Mechanism of action for the pharmacological chaperone AT2101. GCase is a lysosomal hydrolase with optimal stability and activity in the low pH environment of lysosomes. In the near-neutral pH of the endoplasmic reticulum (ER) where GCase initially folds into its tertiary structure, the protein is not as stable as in lysosomes; as a result, a fraction of the synthesized protein is subject to degradation by the ER quality control system. AT2101 binding to GCase in the ER stabilizes GCase, allowing passage through ER quality control and increased trafficking to lysosomes. Once in lysosomes, a combination of factors makes GCase more accessible to its substrates: the reversible nature of the binding and dissociation of AT2101, competition of the natural substrates with AT2101, and the low pH that favors dissociation of AT2101. The net result is an increase in lysosomal GCase activity even in the presence of pharmacological chaperones such as AT2101. Note that the small molecule AT2101 (147 Da) and the enzyme GCase (60 kDa) are not drawn to scale. ERAD = endoplasmic reticulum-associated protein degradation. b , c AT2101 levels were monitored in tissues of 1-month-old Thy1-aSyn mice after a continuous treatment over b 7 or c 3 days followed by a 24‐h washout. All tissues had AT2101 levels above the Ki (inhibition constant of AT2101 towards WT GCase at pH 5.2 = 26 nM) after the 24-h washout, consistent with the need for a washout period (either 4 or 7 days “off”) to provide a period in which GCase is unencumbered by AT2101 inhibition. Bars represent the mean of the group + SEM ( n = 7 mice per group); only values with detectable AT2101 concentration are shown (“n” number in column); duplicate measurements were done for each tissue and mouse; n.d. = not determined owing to insufficient tissue availability. d GCase activity in brain, liver, and spleen from 1- or 5-month-old WT and Thy1-aSyn mice not administered AT2101 (baseline). Interestingly, 5- but not 1-month-old Thy1-aSyn mice had increased GCase activity in the liver compared with WT mice. Furthermore, we found a slight decrease in GCase activity with age in brain and liver, which reached significance in WT mice only. ** p < 0.01 vs WT, ^ p < 0.05 vs 1 month, 2 × 2 analysis of variance, Bonferroni t test, mean + SEM ( n = 6–10 mice per group), measurements done in duplicate for each sample. Fold changes after AT2101 administration are shown in Table . e Quantity of endoglycosidase H (EndoH)‐resistant GCase extracted from subcortex (brain tissue after dissection of cortex and cerebellum, consisting mostly of striatum, hippocampus, and thalamus) of 1-month-old Thy1-aSyn mice administered vehicle or AT2101 (3-on/1-off or 7-on/1-off) monitored via Western blotting, indicative of GCase that has trafficked beyond the ER and reached the mid-Golgi apparatus . To enable a comparison of changes in GCase levels between the 2 regimens, each value was normalized to the mean value of the respective 5 vehicle animals of the regimen. Images of blots for GCase and beta-actin loading control, data as mean + SEM ( n = 5), ** p < 0.01 compared with vehicle; no significant differences between 3 and 7 days administration (Student’s t test)
Techniques Used: Activity Assay, Mouse Assay, Synthesized, Binding Assay, Inhibition, Concentration Assay, Dissection, Western Blot
15) Product Images from "Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation"
Article Title: Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation
Journal: BMC Genomics
doi: 10.1186/1471-2164-12-463
Figure Legend Snippet: HIPPI interacts with P53 in the nucleus and alters the expression of Procaspase1 in cell . A. Co-immunoprecipitation of HIPPI with P53 from whole cell extract of Neuro2A cells. IP was carried out with anti P53 antibody and western blot was done with anti HIPPI antibody ( Upper panel) . The same blot was stripped and reprobed with anti P53 antibody as IP control ( lower panel ). Lane 'In': input of immunoprecipitation, '+Ab': cell extract treated with anti P53 antibody, '-Ab': cell extract treated with IgG only. B. Co-immunoprecipitation of HIPPI with P53 from cytoplasm and nuclear compartment of Neuro2A cells. Cytoplasmic ('CE') and nuclear ('NE') fractions were separately immunoprecipitated with anti P53 antibody and detected with anti HIPPI antibody ( Upper panel ). The lane markings carry their usual meanings as described in A. C. Interaction of HIPPI with P53 in cytoplasm and nuclear compartment of N2AHip1Si cells. D. Western blot analysis for the detection of Procaspase1 expression in parental HeLa cells ('HeLa'), HeLa cells transfected with GFP-Hippi ('Hippi'), p53 knocked down HeLa cells exogenously transfected with GFP-Hippi ('P53SiHi'), HeLa cells transfected with full length p53 ('P53') and HeLa cells co-transfected with full length p53 and GFP-Hippi ('P53+Hippi'). Beta actin was used as loading control. E. Bar diagram represents mean (n = 3) relative expression of Procaspase1 in different cells described in D above. IOD value of the Procaspase1 specific band was normalized with that of beta actin. Normalized IOD of 'HeLa' was considered to be 1.
Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection
Figure Legend Snippet: Relative expression level of some of the genes altered in HD microarray study in presence of HIPPI . Expression of beta actin was taken as control. Error bar represents standard deviation (n = 3). For each gene, expression level in 'Hippi' was compared with expression level in 'HeLa' and that in 'Hip1SiHi' with 'Hippi'. Significance levels of various pairs are indicated in the figure.
Techniques Used: Expressing, Microarray, Standard Deviation
Figure Legend Snippet: Relative expression level of some of the altered genes in microarray study determined by RT-PCR . Expression of beta actin was taken as control. Error bar represents standard deviation (n = 3). For each gene, expression level in 'Hippi' was compared with expression level in 'HeLa' and that in 'Hip1SiHi' with 'Hippi'. Significance levels of various pairs are indicated in the figure.
Techniques Used: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Standard Deviation
16) Product Images from "Next-gen sequencing identifies non-coding variation disrupting miRNA binding sites in neurological disorders"
Article Title: Next-gen sequencing identifies non-coding variation disrupting miRNA binding sites in neurological disorders
Journal: Molecular psychiatry
doi: 10.1038/mp.2017.30
Figure Legend Snippet: rs72727021 is associated with ARHGEF39 expression in the human brain. (A) A multi-tissue eQTL comparison was made for rs72727021 and ARHGEF39 using the GTEx database gene association tool across 44 tissue types. For each tissue is shown: the number of RNA-Seq samples with genotype (‘Samples’) , the effect size (‘Beta’), the significance for that tissue (‘p-value’) and the posterior probability likelihood that an effect exists in each tissue in the cross-tissue meta-analysis (‘post-prob’). Tissues are listed by effect size and those with a post-prob > 0.9 and p-value below p
Techniques Used: Expressing, RNA Sequencing Assay
17) Product Images from "TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration"
Article Title: TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Journal: PLoS Biology
doi: 10.1371/journal.pbio.1001726
Figure Legend Snippet: TJ recruitment of TRAF4 is PIP-binding dependent. (A) Schematic representation of sh-insensitive Flag-tagged WT and mutant TRAF4 constructs used to reintroduce TRAF4 expression in MCF7/shT4 cells. (B) Recruitment of WT and mutant TRAF4 proteins (green) at TJs was analyzed by colocalization with ZO-1 (red). The highlighted overlap (white) between TRAF4 and ZO-1 staining is shown on merge panels and alone on the right panel. While the TRAF4-K313E mutant is still partially colocalized with ZO-1 (middle panels), the TRAF4-K345E mutant does not colocalize anymore with ZO-1 (bottom panels). Scale bar, 10 µm. (C) Quantification of WT and mutant TRAF4 recruitment at TJs. The colocalization index (overlapping area between TRAF4 and ZO-1 staining divided by the TJ length) was measured on 10 microscopic fields. Compared to the WT protein, the colocalization index was reduced by 40% and 78% for K313E and K345E TRAF4 mutants, respectively. (D) Western blot analysis of TRAF4 protein level in parental MCF10A and in TRAF4-silenced cells (MCF10A/shT4) where WT (MCF10A/shT4+TRAF4) and mutant (MCF10A/shT4+TRAF4K345E) TRAF4 expression was restored. The MCF10A/shT4+pBABE cell line represents a control line transduced with the empty vector. Beta-actin was used as a loading control. (E) The presence of TJs was estimated by ZO-1 staining in parental (a) and in TRAF4-silenced cells where the expression of WT (c) and mutant TRAF4 (d) was reintroduced. TRAF4-silenced cell line transduced with the empty vector (b) was used as a control. The PIP-binding–deficient TRAF4-K345E cannot rescue the phenotype induced by TRAF4 silencing on TJs. Left panels, representative confocal image sections of ZO-1 staining (green); right panels, merge with Hoechst staining (blue). Scale bar, 20 µm. (F) TJ quantification in cell lines described in (D) and (E) was performed as described in Figure 1E . n, number of microscopic fields used for the quantification.
Techniques Used: Binding Assay, Mutagenesis, Construct, Expressing, Staining, Western Blot, Transduction, Plasmid Preparation
18) Product Images from "miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population, et al. miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population"
Article Title: miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population, et al. miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population
Journal: Journal of Cellular and Molecular Medicine
doi: 10.1111/jcmm.13901
Figure Legend Snippet: The relative protein levels of gene SYK in cells transfected with mi RNA mimics. (A) THP ‐1 or U937 cells were transfected with 50 nM mimic control or the miR‐129‐2‐3p mimic, respectively. After 48 hours of transfection, cells were harvested and the total protein lysates were analysed for Syk protein levels by Western blotting. Beta‐actin served as the loading control. (B) Quantifications of immunoblotting were expressed as mean ± SD from three independent experiments. ** P
Techniques Used: Transfection, Western Blot
Figure Legend Snippet: The expression levels of miR‐129‐2‐3p and SYK gene in IS patients and control volunteers. (A) miR‐129‐2‐3p expression was detected in the whole blood of 270 ischaemic stroke patients and 270 control volunteers. The relative expression levels were normalized to U6 and then log‐transformed. (B) The expression levels of SYK gene were detected in the whole blood of 60 ischaemic stroke patients and 60 control volunteers. The relative expression levels were normalized to beta‐actin and then log‐transformed. The whiskers of the plots represent the 5‐95 percentiles. Comparison of gene expression between the two groups of cohorts was analysed by the Student's t test
Techniques Used: Expressing, Transformation Assay
Figure Legend Snippet: Relative mRNA levels of the gene SYK in cells transfected with mi RNA mimics. (A and B) THP ‐1 or U937 cells were transfected with 50 nM mimic control or the mi RNA mimics, respectively. After 48 hours of transfection, cells were harvested and the total RNA was extracted. The group of cells treated with mimic control ( NC ) served as the control, and the relative expression of SYK was detected by qRT ‐ PCR , normalized to the mRNA levels of beta‐actin in each group. Quantifications of the mRNA levels of SYK were expressed as mean ± SD from three independent experiments. ** P
Techniques Used: Transfection, Expressing, Quantitative RT-PCR
19) Product Images from "Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins"
Article Title: Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins
Journal: mSphere
doi: 10.1128/mSphere.00234-17
Figure Legend Snippet: Schematic summary of the mechanisms by which tick-borne Phlebovirus NSs proteins inhibit the canonical IFN induction and signaling pathways. Following bunyavirus infection, the generation of ssRNA with uncapped 5′ triphosphate ends during uncoating, transcription, or replication results in ssRNA binding to the RNA helicase RIG-I. RIG-I is activated in turn, and exposure of its two associated CARD domains allows the recruitment of the adaptor MAVS through two CARD-CARD interactions. Activation of MAVS leads to the subsequent activation of kinases such as TBK1 and/or IKKε, which leads to concomitant phosphorylation of IRF3 at specific serine residues. (A) Phosphorylation of IRF3 leads to its dimerization and translocation to the nucleus, where IRF3 dimers ultimately stimulate transcription of genes under the control of the IFN-α/β promoter, resulting in the production of IFN-α/β. (B) Signal transduction of type I IFNs initiates through the binding of secreted IFN-α/β to type I IFN receptors (a heterodimer of IFNAR1 and IFNAR2) and the activation of multiple downstream signaling pathways. Signal transduction of type I, II, and III IFN initiates through the binding of secreted IFN to the respective IFN receptors and the activation of multiple downstream signaling pathways. Type I and type III IFN signaling pathways are mainly mediated via STAT1-STAT2 heterodimers. Receptor-associated kinases JAK1 and TYK2 become activated and phosphorylate STAT1 and STAT2, respectively. Phosphorylated STATs can heterodimerize and recruit IRF9 for the assembly of the heterotrimer complex ISGF3. ISGF3 translocates to the nucleus and binds to ISRE, leading to the induction of many IFN-stimulated genes (ISG). Type III IFN signaling is more commonly associated with signaling by homodimerization of STAT1, which can translocate to the nucleus and activate GAS promoters, also initiating ISG transcription. Tick-borne Phlebovirus NSs proteins have evolved several countermeasures to block the IFN pathway. SFTSV NSs (red) directly interacts with and sequesters TBK1 and STAT2 into inclusion bodies to spatially isolate these elements. Additionally, through its interaction with TBK1 and STAT2, SFTSV NSs can also indirectly sequester IKKε, IRF-3, and STAT1 into the inclusion bodies. An interaction between SFTSV NSs and TRIM-25 also facilitates the spatial isolation of RIG-I, in an indirect manner. HRTV NSs (yellow) can also block IFN induction through a direct interaction with TBK1 and block IFN signaling by a direct interaction with STAT2. However, as HRTV NSs does not form inclusion bodies as SFTSV NSs does, its mechanism of antagonism is different from that of SFTSV NSs. UUKV NSs (green) blocks IFN induction only, targeting MAVS. For references, see the text. Dashed red lines indicate indirect interactions. Solid red lines indicate direct interactions. For references, see the text. CARD, caspase recruitment domain; GAS, gamma-activated sequence; IFNAR, interferon-α/β receptor; IFNGR1, interferon gamma receptor 1; IκB, inhibitor of kappa B; IKK, inhibitor of nuclear factor kappa-B kinase subunit alpha; IKK β, inhibitor of nuclear factor kappa-B kinase subunit beta; IKKε, IκB kinase-ε; IRF, IFN-regulatory factor; ISGF3, IFN-stimulated gene factor 3; ISGs, IFN-stimulated genes; ISRE, IFN-stimulated response elements; JAK1, Janus kinase 1; MAVS, mitochondrial antiviral signalling protein; NFκB, nuclear factor kappa-light-chain enhancer of activated B cells; RIG-I, retinoic acid-inducible gene I; STAT, signal transducer and activator of transcription; TBK1, TANK-binding kinase 1; TYK2, tyrosine kinase 2.
Techniques Used: Infection, Binding Assay, Activation Assay, Translocation Assay, Transduction, Blocking Assay, Isolation, Sequencing
20) Product Images from "The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592"
Article Title: The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592
Journal: Cell Cycle
doi: 10.1080/15384101.2018.1480216
![Phosphorylation status of SAMHD1 during cell cycle progression in normal human fibroblasts (A) and transformed cells (B). A . Cycling lung fibroblasts were synchronized by double thymidine block and released. Confluent fibroblasts were maintained for 48 h in medium with 10 or 0.1% serum. The top panel shows the cell cycle profiles at the indicated times after release and at confluence. The lower panels show the expression levels of SAMHD1 detected by an antibody directed against phosphoT592 (pSAMHD1) and a mouse monoclonal antibody recognizing both the phosphorylated and non- phosphorylated forms of SAMHD1 (SAMHD1). Cyclin E, A2 and B were used as cell cycle markers and beta-actin as a loading control. B . Whole cell extracts of HeLa cells synchronized by double thymidine block were analyzed for expression of SAMHD1 with the same mouse monoclonal recognizing both forms of SAMHD1. The phosphorylated form appears as a slower migrating band (empty arrow) compared to the non-phosphorylated form (black arrow). The cell cycle markers cyclin E, cyclin A and histone H3-S10-phosphorylation are those analyzed in the same experiment and data for these markers was published in Figure 2B of Kara et al [ 25 ].](https://storage.googleapis.com/bioz_article_images/PMC6110608/kccy-17-09-1480216-g002.jpg "... B were used as cell cycle markers and beta-actin as a loading control. B . Whole cell ...")
Figure Legend Snippet: Phosphorylation status of SAMHD1 during cell cycle progression in normal human fibroblasts (A) and transformed cells (B). A . Cycling lung fibroblasts were synchronized by double thymidine block and released. Confluent fibroblasts were maintained for 48 h in medium with 10 or 0.1% serum. The top panel shows the cell cycle profiles at the indicated times after release and at confluence. The lower panels show the expression levels of SAMHD1 detected by an antibody directed against phosphoT592 (pSAMHD1) and a mouse monoclonal antibody recognizing both the phosphorylated and non- phosphorylated forms of SAMHD1 (SAMHD1). Cyclin E, A2 and B were used as cell cycle markers and beta-actin as a loading control. B . Whole cell extracts of HeLa cells synchronized by double thymidine block were analyzed for expression of SAMHD1 with the same mouse monoclonal recognizing both forms of SAMHD1. The phosphorylated form appears as a slower migrating band (empty arrow) compared to the non-phosphorylated form (black arrow). The cell cycle markers cyclin E, cyclin A and histone H3-S10-phosphorylation are those analyzed in the same experiment and data for these markers was published in Figure 2B of Kara et al [ 25 ].
Techniques Used: Transformation Assay, Blocking Assay, Expressing
Figure Legend Snippet: Kinetics of SAMHD1 dephosphorylation during mitotic exit in hRPE-1 cells. A . hRPE-1 cells were synchronized in prometaphase by 16 h nocodazole treatment (100 ng/ml), mitotic cells were detached by gentle shake-off and seeded in fresh medium. Samples were collected without nocodazole synchronization (A), immediately after shake-off (M) and after 2 h in fresh medium (early G1) and analyzed by immunoblotting. SAMHD1 was detected by a mouse monoclonal antibody recognizing both the phosphorylated (empty arrow) and the non-phosphorylated form (black arrow) as indicated by a band shift. Cyclin A2, cyclin B and R2, the S-phase induced small subunit of ribonucleotide reductase, are used as cell cycle markers. GAPDH: loading control. B . Schematic diagram of the protocol used to synchronize mitotic exit in hRPE-1 cells. Samples were collected in prometaphase and during mitotic exit at the indicated time points after RO3306 addition. The proteasome inhibitor MG132 was present 15 min before RO3306 addition and during mitotic exit. C . hRPE-1 cells were synchronized as in B and whole cell extracts were immunoblotted for cyclin B, securin, phospho-Histone H3(Ser10) (pH3S10), pThr-CDK substrates, pSer-CDK substrates and beta-actin (loading control). In the same experiment SAMHD1 phosphorylation was analyzed as in A. by electrophoresis in a phosgel using the antibody against phospho T592 (pSAMHD1) after pre-treatment in the presence (+) or absence (-) of lambda phosphatase (PP). Empty arrow: pSAMHD1, black arrow non-phosphorylated SAMHD1. Decay of protein phosphorylation was evaluated from the relative intensities of pSAMHD1, pThr-CDK substrates and pH3S10 normalized for beta-actin and the level of phosphorylation at 0 min, taken as 100%. Bars: Mean ± standard error for n = 4 values. D . hRPE-1 cells were synchronized as in B. After RO3306 addition one set of cultures was treated with proteasome inhibitor MG132 (RO3306 +MG132). Samples were collected at 0–5-15–30 min during mitotic exit and immunoblotted for cyclin B, securin, pThr-CDK substrates, pSer-CDK substrates and beta-actin (loading control). SAMHD1 was analyzed as described in C. *non-specific band
Techniques Used: De-Phosphorylation Assay, Electrophoretic Mobility Shift Assay, Electrophoresis
Figure Legend Snippet: dNTP pools in different phases of the cell cycle in SAMHD1-KO and parental THP-1 cells. A . cell proliferation curve and cell cycle analysis of THP-1 cells knock-out for SAMHD1 (KO – dashed line) and the parental control (wt – solid line). Bars: Mean ± standard error for n = 3 values. B . Representative image of the cell cycle distribution of THP1 cells before (A = asynchronous population) and after centrifugal elutriation (G1, S and G2/M enriched sub-populations). C . Immunoblotting for SAMHD1, RNR subunits and cell cycle markers in each enriched sub-population of wt and KO THP-1 cells. Beta-actin: loading control. SAMHD1 was detected using a mouse polyclonal antibody D . Phosphorylated SAMHD1 was detected after electrophoresis in a phosgel using an antibody directed against pT592 (α-pSAMHD1) and a rabbit polyclonal antibody recognizing both the phosphorylated and the non-phosphorylated form (α-SAMHD1). Before electrophoresis, parallel samples were treated with (+) or without (-) lambda phosphatase (lambda PP). E . Comparison of dNTP pool sizes in wt and KO THP-1 cells in the indicated phases of the cell cycle. In the individual phases the level of each dNTP was calculated as detailed in Experimental procedures. The fold increase of each dNTP in KO cells relative to the wt is reported for each phase of the cycle. Bars: Mean ± standard deviation for n = 6 values. F. The composition of the dNTP pools in each phase of the cycle for wt and KO cells, evaluated from the ratios between pmoles of dATP, dTTP or dCTP and pmoles of dGTP under each condition.
Techniques Used: Gene Knockout, Cell Cycle Assay, Knock-Out, Electrophoresis, Standard Deviation
Figure Legend Snippet: Turn-over of SAMDH1 in proliferating U2OS cells. A . The expression of GFP-tagged SAMHD1 was induced in U2OS cells stably transfected with a tetracyclin-inducible vector by treatment with different doses of tetracycline (tet) for 24 h and analyzed by immunoblotting. The lower panel reports a representative immunoblot of endogenous SAMHD1 (Endo-SAMHD1) and ectopically expressed GFP-SAMHD1. B . After tet removal the decline of the induced mRNA was followed by RT-PCR. The level of mRNA is reported as fold increase relative to that of not induced transfected cells (dashed line). C . The amount of GFP-SAMHD1 was determined by immunoblotting using antibodies against SAMHD1 and GFP. Representative immunoblots of total GFP-SAMHD1 (α-SAMHD1) and phosphorylated GFP-SAMHD1 (α-pSAMHD1) in samples induced for 24 h with 1 µg/ml tet and chased in the absence of tet for the indicated times. Beta actin: loading control. NI = not induced. D . Densitometric analysis was performed for GFP-SAMHD1, normalized for beta-actin and expressed as percentage relative to the protein level at 0 time. Bars in A.B. and D: Mean ± standard error, n = 3.
Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot
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After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) Western Blot:Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand Article Snippet: Paragraph title: Western blotting ... Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells Article Snippet: Paragraph title: Protein Isolation and Western Blots. ... Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line Article Snippet: Paragraph title: Western blot analysis ... PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 ( Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease Article Snippet: AGS3 protein detection was determined using standard Western blot techniques [ , , ]. .. Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology Article Snippet: Paragraph title: 4.5. Protein Isolation and Western Blotting ... The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a Article Title: Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways Article Snippet: Paragraph title: 4.8. Western Blotting ... The blots were stripped with buffer (2.5% SDS, 0.7% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and incubated with the Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS Article Snippet: Paragraph title: Western blot assay ... The Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication Article Snippet: Paragraph title: 2.1.6. Western blot analysis ... The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and Article Title: Transcriptional coactivator PGC-1α contains a novel CBP80-binding motif that orchestrates efficient target gene expression Article Snippet: Paragraph title: Western blotting ... Proteins were detected using the following antibodies: anti-PGC-1β (Bethyl Laboratories, A302-273A), anti-PGC-1α (Novus, NBP1-04676), anti-Flag (Sigma-Aldrich, A8592), Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia Article Snippet: Paragraph title: Western blotting ... Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells Article Snippet: Paragraph title: Western Blot Analysis ... Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and Article Title: Expression of Extracellular Superoxide Dismutase Protein in Diabetes Article Snippet: Paragraph title: Western blot analysis ... The membranes were stripped and reblotted with Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication Article Snippet: The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12-DN) constructs were kindly provided by Dr. Adi Kimchi (Israel) [ ]. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and Article Title: Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling Article Snippet: Paragraph title: Western blot analysis ... Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1 Article Snippet: Paragraph title: Distal lung protein extraction and Western blot analysis ... Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells Article Snippet: Paragraph title: Western Blotting Analysis of CREB, Phospho-CREB, ERK, Phospho-ERK and β-actin Proteins ... The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Protease Inhibitor:Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication Article Snippet: Briefly, protein extracts were prepared by cell lysis in RIPA buffer (Sigma-Aldrich) supplemented with a mammalian protease inhibitor cocktail (Sigma-Aldrich). .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and Article Title: Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling Article Snippet: Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), monoclonal mouse anti-β-actin (1∶15,000; Sigma), polyclonal goat anti-ATR N19 (1∶1,000; Santa Cruz), monoclonal mouse anti-Chk1 (1∶1,000; Santa Cruz), monoclonal mouse anti-HA (F7) (1∶3,000; Santa Cruz), monoclonal mouse anti-RPA32 (9H8) (1∶1,000; Genetex), monoclonal rabbit anti-phospho-Chk1 S345 (1∶5,000; Cell Signaling), polyclonal rabbit anti-phospho-RPA S33 (1∶3,000; Bethyl), and polyclonal rabbit anti-phospho-RPA S4/S8 (1∶3,000; Bethyl). .. Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), Cell Culture:Article Title: Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study Article Snippet: Proteins from normal blood MNC cultured with cisplatin and phytohemagglutinin (PHA), and untreated MNC probed with antibodies on the same membrane served as positive and negative controls respectively. .. To control for protein loading, each membrane was re-probed with a mouse monoclonal antibody to Generated:Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor Article Snippet: Supernatants from TIMP-3/HEK or HEK293 cells were loaded onto an acrylamide gel for electrophoresis. .. After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) other:Article Title: Insulin-like growth factor receptor signaling in breast tumor epithelium protects cells from endoplasmic reticulum stress and regulates the tumor microenvironment Article Snippet: Imaging:Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells Article Snippet: Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or Protein Concentration:Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line Article Snippet: Protein concentration was determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 ( Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS Article Snippet: The protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). .. The Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication Article Snippet: Protein concentration was determined using the Pierce BCA protein assay kit (Life Technologies), according to the manufacturer’s instructions. .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia Article Snippet: The protein concentration of each sample was determined using the DC Protein Assay kit (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells Article Snippet: The protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Binding Assay:Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia Article Snippet: Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), Molecular Weight:Article Title: Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways Article Snippet: After denaturing in Laemmli buffer (catalog #161-0737, Bio-Rad, Hercules, CA, USA), equal amounts of protein (10–20 μg) were loaded onto 4–15% or 4–20% gradient precast gels (Bio-Rad), depending on the molecular weight of the target protein. .. The blots were stripped with buffer (2.5% SDS, 0.7% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and incubated with the DC Protein Assay:Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia Article Snippet: The protein concentration of each sample was determined using the DC Protein Assay kit (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), Isolation:Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand Article Snippet: Proteins were extracted, and the soluble cytosolic fraction was isolated according to our previous study [ ]. .. Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells Article Snippet: Paragraph title: Protein Isolation and Western Blots. ... Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease Article Snippet: SD and PCK rat kidney lysates at 8, 16, and 24 weeks of age, and human and rat urine exosome protein lysates were isolated using 1X RIPA buffer containing protease (Roche) and phosphatase inhibitors (Pierce, Rockford, IL). .. Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology Article Snippet: Paragraph title: 4.5. Protein Isolation and Western Blotting ... The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a Protein Extraction:Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1 Article Snippet: Paragraph title: Distal lung protein extraction and Western blot analysis ... Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and Construct:Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication Article Snippet: The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12-DN) constructs were kindly provided by Dr. Adi Kimchi (Israel) [ ]. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and Polyacrylamide Gel Electrophoresis:Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand Article Snippet: Samples containing equivalent amounts of protein were loaded onto 10% to 15% acrylamide denaturing gels (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE). .. Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and Staining:Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication Article Snippet: The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich). .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and SDS Page:Article Title: P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand Article Snippet: Samples containing equivalent amounts of protein were loaded onto 10% to 15% acrylamide denaturing gels (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE). .. Membranes were probed overnight at 4°C with antibodies against Fas (sc-736), FasL (sc-6237), P2X7 (sc-25698), High mobility group box 1 (HMGB1, sc-26351), caspase-8 (sc-7890) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); CD3 (Abcam Cambridge, UK, Catalog no: ab5690); FADD (Epitomics, Inc., Burlingame, CA, USA, Catalog no: 2988–1); and Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells Article Snippet: The whole-cell proteins (35 μ g/lane) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. .. Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line Article Snippet: Approximately 50 μg of lysate was fractioned in 15% SDS-PAGE gels that were subsequently transferred to PVDF Immobilon-P membranes (IPVH00010, Millipore-Merck). .. PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 ( Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology Article Snippet: Twenty micrograms (20 μg) of protein lysates were separated in 1.5 mm thick 12% SDS-PAGE gels. .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS Article Snippet: Proteins (30 µ g) were separated on 10% SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. .. The Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia Article Snippet: The proteins were resolved by SDS-PAGE (8%) using a Mini-Protean system (Bio-Rad) and depending on the epitope to be analyzed, 30–50 μg of total protein was loaded into each lane in loading buffer containing: 0.062 M Tris (pH 6.8), 10% glycerol, 5% β-mercaptoethanol, 7.5 mM EDTA, 2% SDS, and 0.002% bromophenol blue. .. Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells Article Snippet: Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). .. Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and Article Title: Expression of Extracellular Superoxide Dismutase Protein in Diabetes Article Snippet: Proteins from tissue were separated by SDS-PAGE using NuPAGE 4% to 12% bis-Tris gels (Invitrogen, Carlsbad, CA, USA, NP0335Box) and then transferred to Immobilon-P membrane. .. The membranes were stripped and reblotted with Article Title: Herpes Simplex Virus Type 1 Single Strand DNA Binding Protein and Helicase/Primase Complex Disable Cellular ATR Signaling Article Snippet: Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. .. Primary antibodies used include polyclonal rabbit anti-ATRIP 403 (rATRIP 403) (1∶3,000) , monoclonal mouse anti-ICP4 (1∶10,000; US Biologics), Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1 Article Snippet: Lungs were removed from thorax and homogenized for 3 min in ice-cold lysis RIPA buffer (pH 8) containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and protease inhibitors. .. Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and Article Title: Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells Article Snippet: Cell lysate (30 µg) was separated on 10% SDS-PAGE and transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA) at 25 volts overnight at 4°C. .. The membranes were blocked at 4°C in PBST blocking buffer (1% non-fat dried milk in PBS containing 0.1% Tween-20) for 8 h. Blots were incubated with the appropriate antibodies overnight at 4°C: anti-CREB (1∶1000), anti-phospho-CREB (Ser-133) (1∶1000), anti-ERK (1∶1000 ), anti-phospho-ERK (1∶1000 ) (Cell Signaling Technology, Inc.), and Plasmid Preparation:Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication Article Snippet: hATG5 and hATG16L1 sequences were cloned into peGFP-C1 plasmid (Clontech) to form pGFP-ATG5 and pGFP-ATG16L1, respectively. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and Software:Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells Article Snippet: Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or Article Title: Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line Article Snippet: PACRG was detected using rabbit anti-PACRG clone MC1290 diluted 1:1000 . β-Actin was used as a loading control and was detected by mouse anti-β-Actin ascites clone AC-15 diluted 1:10000 ( Article Title: Urinary exosomal expression of activator of G protein signaling 3 in polycystic kidney disease Article Snippet: Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology Article Snippet: The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication Article Snippet: The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and Article Title: Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia Article Snippet: Subsequently, the membranes were blocked for 1 h with 5% non-fat powdered milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and then they were incubated overnight at 4 °C with the appropriate primary antibody: Akt (1:1,000; Cell Signaling no. 9272); phospho-AktSer473 (1:500; Cell Signaling no. 9271), phospho-AktThr308 (1:1,000; clone C31E5E, Cell Signaling no. 2965), β-catenin (1:800; BD Transduction Laboratories no. 610153), β-tubulin (1:1,000; clone TUB 2.1, Sigma no. T4026), Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1 Article Snippet: Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and Dominant Negative Mutation:Article Title: LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication Article Snippet: The Flag-tagged ATG12 (pATG12) and its dominant-negative derivative pATG12ΔG140 (ATG12-DN) constructs were kindly provided by Dr. Adi Kimchi (Israel) [ ]. .. Rabbit polyclonal anti-LC3, rabbit polyclonal anti-ATG5 (used for western blot), mouse monoclonal anti-Flag, and In Vitro:Article Title: Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study Article Snippet: To control for protein loading, each membrane was re-probed with a mouse monoclonal antibody to Radio Immunoprecipitation:Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS Article Snippet: Briefly, radioimmunoprecipitation assay buffer (Qiagen, Inc., Valencia, CA, USA) was used to extract total proteins from Huh7 cells. .. The Concentration Assay:Article Title: Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology Article Snippet: The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a reference protein (Sigma-Aldrich, St. Louis, MO, USA, A5441, 1:5000). .. The following antibodies and dilutions were used: anti-GLO1 (Sigma-Aldrich, St. Louis, MO, USA, SAB4200193, 1:4000) and anti-β-actin as a Lysis:Article Title: Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells Article Snippet: Proteins were extracted from harvested cells using radioimmunoprecipitation lysis buffer supplemented with complete protease inhibitors, and protein concentrations were determined using the BCA Protein Assay Kit. .. Membranes were incubated in 5% Blotting-Grade Blocker (catalog #170-6404; Bio-Rad) at room temperature for 2 hours, and then with primary antibodies, anti-NRF2 antibody (1:1000 dilution, catalog #ab62352; Abcam, Cambridge, MA), or Article Title: Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication Article Snippet: Briefly, protein extracts were prepared by cell lysis in RIPA buffer (Sigma-Aldrich) supplemented with a mammalian protease inhibitor cocktail (Sigma-Aldrich). .. The membranes were probed with the following primary antibodies; polyclonal rabbit anti-TLR-4 (M-300) (sc-30002, Santa Cruz), polyclonal rabbit anti-GLT-1 (pab0037, Covalab), polyclonal rabbit anti-GLAST (pab0036-P, Covalab), polyclonal rabbit anti-connexin 4371–0700, Life technologies), monoclonal mouse anti-Na+/K+-ATPase (A276, Sigma-Aldrich), and Article Title: Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells Article Snippet: Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). .. Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and Article Title: ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1 Article Snippet: Lungs were removed from thorax and homogenized for 3 min in ice-cold lysis RIPA buffer (pH 8) containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and protease inhibitors. .. Samples of protein extracts (100–200 µg/sample, the amount necessary to detect minor bands of ENaC proteins) were separated by SDS-PAGE on 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for ENaC subunits and β-actin by using previously characterized rabbit polyclonal anti rat α-, β- and γ-ENaC antibodies (dilution 1:2000) (Duc et al, ), and T-Test:Article Title: Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor Article Snippet: After electrophoretic separation, proteins were blotted onto a PVDF membrane using the Trans-Blot Turbo transfer system (Biorad) and detected by the following antibodies: anti-TIMP-3 (AB6000, Millipore), anti-TIMP-1 (generated as previously described ), anti-TIMP-2 (generated as previously described ), anti-SPARC (number 5031, Thermo Fisher), anti-MIF (clone FL-115, Santa Cruz) anti-APP (clone 22C11, Millipore), anti-MMP-1 (clone 2A7.2, Millipore) Activity Assay:Article Title: Reduced PKC α Activity Induces Senescent Phenotype in Erythrocytes Article Snippet: PMA (P 8139), 4α -phorbol 12,13-didecanoate (4α PDD) (P 8014), phenyl methyl sulfonyl fluoride (PMSF) (P 7626), anti-band 3 N-terminus monoclonal antibody (B 9277), and |
