Western Blot Analysis of C. elegans Proteins

2018 ◽  
pp. 213-225 ◽  
Author(s):  
Dae-Eun Jeong ◽  
Yujin Lee ◽  
Seung-Jae V. Lee
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
C Elegans

scholarly journals Localization, Regulation and Possible Consequences of Apoptotic Protease-Activating Factor-1 (Apaf-1) Expression in Granulosa Cells of the Mouse Ovary

Endocrinology ◽  
1999 ◽  
Vol 140 (6) ◽  
pp. 2641-2644 ◽  
Author(s):  
Rodolfo Robles ◽  
Xiao-Jing Tao ◽  
Alexander M. Trbovich ◽  
Daniel V. Maravei ◽  
Ravit Nahum ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
C Elegans

Abstract The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bc1-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals Protocol for determining protein cysteine thiol redox status using western blot analysis

2021 ◽  
Vol 2 (2) ◽  
pp. 100566
Author(s):  
Bikram Datt Pant ◽  
Sunhee Oh ◽  
Kirankumar S. Mysore
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Redox Status ◽  
Thiol Redox

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

scholarly journals OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Alessandro Bressan ◽  
Francesca Bozzo ◽  
Carlo Alberto Maggi ◽  
Monica Binaschi
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Tandem Repeat ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Ovarian Cancer Cells ◽  
Repeat Region ◽  
Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

scholarly journals DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jiechao Yang ◽  
Liang Zhou ◽  
Yanping Zhang ◽  
Juan Zheng ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Laryngeal Squamous Cell Carcinoma ◽  
Poor Overall Survival

Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.

scholarly journals Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

Reproduction ◽  
2003 ◽  
pp. 495-507 ◽  
Author(s):  
SA Joshi ◽  
S Shaikh ◽  
S Ranpura ◽  
VV Khole
Keyword(s):  
Western Blot ◽  
Postnatal Development ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antiserum ◽  
Cognate Gene ◽  
Specific Proteins ◽  
Androgen Regulation ◽  
Dose Dependent

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.

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