scholarly journals Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

2021 ◽  
Author(s):  
Angelica Kirsten Florentinus
Keyword(s):  
Western Blot ◽  
Affinity Chromatography ◽  
Fc Receptor ◽  
Actin Binding ◽  
Annexin Ii ◽  
Receptor Complex ◽  
Cell Staining ◽  
Crude Extracts ◽  
Annexin I

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.

scholarly journals Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

2021 ◽  
Author(s):  
Angelica Kirsten Florentinus
Keyword(s):  
Western Blot ◽  
Affinity Chromatography ◽  
Fc Receptor ◽  
Actin Binding ◽  
Annexin Ii ◽  
Receptor Complex ◽  
Cell Staining ◽  
Crude Extracts ◽  
Annexin I

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

scholarly journals Protein kinase C-dependent phosphorylation of annexins I and II in mesangial cells

1993 ◽  
Vol 292 (1) ◽  
pp. 63-68 ◽  
Author(s):  
J P Oudinet ◽  
F Russo-Marie ◽  
J C Cavadore ◽  
B Rothhut
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mesangial Cells ◽  
Serine Phosphorylation ◽  
Annexin Ii ◽  
Kinase C ◽  
Annexin I ◽  
Phosphopeptide Mapping

In this study we describe the phosphorylation of annexins from cultured rat mesangial cells by protein kinase C (PKC) both in vitro and in vivo. Annexins I and II were detected either by Western-blot analysis or by immunoprecipitation using specific antibodies. In the presence of [gamma-32P]ATP, cytosolic annexin I and annexin II were phosphorylated in vitro only when Ca2+ and phospholipids were added, but not in the presence of phospholipids alone. Annexin I was shown to be a better substrate than annexin II. In experiments in vivo performed on 32P-labelled mesangial cells, the addition of two well-known activators of PKC, namely angiotensin II (AII) and phorbol myristate acetate (PMA), increased preferentially the phosphorylation of annexin I. Annexin II was phosphorylated to a much lesser extent after AII treatment. Phosphoamino acid analysis of annexins, either by two-dimensional chromatography or by using a specific antiphosphotyrosine antibody, revealed only phosphoserine in these experiments in vivo. The addition of AII to mesangial cells increased serine phosphorylation of annexin I and annexin II, whereas PMA only increased serine phosphorylation of annexin I. V8-protease phosphopeptide mapping of annexin I that was phosphorylated both in vitro and in vivo by PKC from mesangial cells shows similar phosphopeptides.

Changes in annexin I and II levels during the postnatal development of rat pancreatic islets

1994 ◽  
Vol 107 (8) ◽  
pp. 2117-2125 ◽  
Author(s):  
M. Ohnishi ◽  
M. Tokuda ◽  
T. Masaki ◽  
T. Fujimura ◽  
Y. Tai ◽  
...  
Keyword(s):  
Western Blot ◽  
Pancreatic Islets ◽  
Postnatal Development ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Patterns ◽  
Annexin Ii ◽  
Developmental Study ◽  
Rat Pancreatic Islets ◽  
Annexin I

The expression patterns and the dynamic changes in content of both annexin I and annexin II in the rat pancreatic islets during postnatal development were investigated by both western blot analysis and immunohistochemistry. Immunohistochemical methods clearly demonstrated the presence of annexins I and II exclusively in pancreatic islets, while exocrine tissues were not stained by anti-annexin antibodies. Pancreatic islets were diffusely stained with no specific differences in distribution between different cell types. The expression of annexin I in pancreatic islets gradually increased with postnatal development. A developmental study of annexins I and II by western blot analysis essentially supported the results obtained by immunohistochemistry. In addition, the increasing expression of two protein tyrosine kinases, epidermal growth factor-receptor/kinase and pp60src, which phosphorylate annexin I and annexin II, respectively, and of protein kinase C, which phosphorylates both proteins, was also shown during postnatal development in rat pancreatic islets. Thus, a relationship between the expression of annexins I and II and the maturation of islet cell function is suggested.

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.

Anticancer Potential of Calli Versus Seedling Extracts Derived from Rosmarinus officinalis and Coleus hybridus

2020 ◽  
Vol 21 (14) ◽  
pp. 1528-1538
Author(s):  
Sarah Albogami ◽  
Hadeer Darwish ◽  
Hala M. Abdelmigid ◽  
Saqer Alotaibi ◽  
Ahmed Nour El-Deen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Transcriptional Profiling ◽  
Significant Inhibition ◽  
Rosmarinus Officinalis ◽  
Crude Extracts ◽  
Incidence And Mortality ◽  
Mcf 7

Background: In Saudi Arabia, the incidence and mortality rates of breast cancer are high. Although current treatments are effective, breast cancer cells develop resistance to these treatments. Numerous studies have demonstrated that active compounds in plant extracts, such as the phenolic compound Rosmarinic Acid (RA), exert anti-cancer effects. Objective: We investigated the anticancer properties of methanolic crude extracts of seedlings and calli of Rosmarinus officinalis and Coleus hybridus, two Lamiaceae species. Methods: MCF-7 human breast cancer cells were treated with methanolic crude extracts obtained from plant calli and seedlings generated in vitro, and cell proliferation was evaluated. Transcriptional profiling of the seedling and callus tissues was also conducted. Results: The mRNA expression levels of RA genes were higher in C. hybridus seedlings than in R. officinalis seedlings, as well as in C. hybridus calli than in R. officinalis calli, except for TAT and C4H. In addition, seedling and callus extracts of both R. officinalis and C. hybridus showed anti-proliferative effects against MCF-7 cells after 24 or 48 h of treatment. Discussion: At a low concentration of 10 μg/mL, C. hybridus calli and seedling extracts showed the most significant anti-proliferative effects after 24 and 48 h of exposure (p < 0.01); controls (doxorubicin) also showed significant inhibition, but lesser than that observed with C. hybridus (p < 0.05). Results with R. officinalis callus and seedling extracts did not significantly differ from those with untreated cells. Conclusion: Methanolic extracts of R. officinalis and C. hybridus are potentially valuable options for breast cancer treatment.

Mixed Mode Chromatography: A Novel Way Toward New Selectivity

2018 ◽  
Vol 20 (1) ◽  
pp. 14-21 ◽  
Author(s):  
Xavier Santarelli ◽  
Charlotte Cabanne
Keyword(s):  
Salt Tolerance ◽  
Affinity Chromatography ◽  
Mixed Mode ◽  
Ionic Groups ◽  
Crude Extracts ◽  
The Way

Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.

Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

2019 ◽  
Vol 16 (3) ◽  
pp. 175-180
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan
Keyword(s):  
Biological Activity ◽  
Botulinum Toxin ◽  
Cell Membrane ◽  
Western Blot ◽  
Biological Function ◽  
C6 Cells ◽  
Green Fluorescence ◽  
Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

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