Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation

2015 ◽  
pp. 267-277 ◽  
Author(s):  
Takahiro Horinouchi ◽  
Koji Terada ◽  
Tsunehito Higashi ◽  
Soichi Miwa
Keyword(s):  
Protein Phosphorylation ◽  
Western Blotting ◽  
Western Blotting Analysis ◽  
Blotting Analysis

scholarly journals Analysis of the physical activity effects and measurement of pro-inflammatory cytokines in irradiated lungs in rats

2012 ◽  
Vol 27 (3) ◽  
pp. 223-230 ◽  
Author(s):  
Renata Cristiane Gennari Bianchi ◽  
Eduardo Rochete Ropelle ◽  
Carlos Kiyoshi Katashima ◽  
José Barreto Campello Carvalheira ◽  
Luiz Roberto Lopes ◽  
...  
Keyword(s):  
Physical Activity ◽  
Western Blotting ◽  
Inflammatory Cytokines ◽  
Lower Lobe ◽  
Whole Body ◽  
Control Group ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Tnf Α ◽  
Pro Inflammatory Cytokines

PURPOSE: To study if the pre-radiotherapy physical activity has radio-protective elements, by measuring the radio-induced activation of pro-inflammatory cytokines as interleukin-6 (il-6), transforming growth factor -β (tgf -β), tumor necrosis factor -α (tnf-α) and protein beta kinase β (ikkβ), through western blotting analysis. METHODS: A randomized study with 28 Wistar hannover rats, males, with a mean age of 90 days and weighing about 200 grams. The animals were divided into three groups: (GI, GII and GIII). GIII group were submitted to swimming for eight weeks (zero load, three times a week, about 30 minutes). Then, the groups (except the control group) were submitted to irradiation by cobalt therapy, single dose of 3.5 gray in the whole body. All animals were sacrificed by overdose of pentobarbital, according to the time for analysis of cytokines, and then a fragment of the lower lobe of the right lung went to western blotting analysis. RESULTS: The cytokines IKK β, TNF-α and IL-6 induced by radiation in the lung were lower in the exercised animals. However, exercise did not alter the radiation-induced increase in tgf-β. CONCLUSION: The results show a lower response in relation to inflammatory cytokines in the group that practiced the exercise pre-radiotherapy, showing that exercise can protect tissues from tissue damage due to irradiation.

scholarly journals Mechanisms Involved in the Anti-Tumor Effects of Toosendanin in Glioma Cells

2021 ◽  
Author(s):  
haiyan huang ◽  
Chaochao Zhang ◽  
Haijun Gao ◽  
Ziqiang Liu ◽  
Jiacheng Lai ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Wound Healing ◽  
Western Blotting ◽  
Colony Formation ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Antitumor Effects ◽  
Western Blotting Analysis ◽  
Blotting Analysis

Abstract Background: Toosendanin (TSN) is a triterpenoid compound mainly used as an ascaris repellant. Recent studies have shown that it possesses antitumor effects in many types of tumor cells. However, the effects of TSN on glioma cells have rarely been reported. Methods: Different assays were performed to investigate the effects of TSN on the different glioma cell lines including U87MG and LN18. The assays included colony formation, wound healing, and transwell assays. Furthermore, Hoechst 3342 staining, flow cytometry, and western blotting analysis were performed to investigate the apoptotic activities of TSN. Finally, the results were confirmed using a xenograft tumor model that comprised of nude mice. Results: In vitro, the CCK-8 and colony formation assays showed that TSN effectively inhibited glioma cell proliferation. Moreover, the inhibitory effects on glioma cell migration and invasion were demonstrated through the wound healing and transwell assays, respectively. Hoechst 33342 staining, flow cytometry, and western blotting assays demonstrated the significant effect of TSN in the apoptosis induction of glioma cells. Furthermore, the anti-glioma effect of TSN was exerted through the inhibition of the PI3K/Akt/mTOR signaling pathways as demonstrated by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model confirmed the suppressive effect of TSN on tumor growth in vivo. Conclusion: Our results suggest that TSN is a promising chemotherapeutic drug for patients with glioma.

scholarly journals So-Ochim-Tang-Gamibang, a Traditional Herbal Formula, Ameliorates Depression by Regulating Hyperactive Glucocorticoid Signaling In Vitro and In Vivo

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Mirim Jin ◽  
Sun Young Park ◽  
Hye Jin Choi ◽  
Younmin Shin ◽  
Eunho Chun ◽  
...  
Keyword(s):  
Western Blotting ◽  
Hpa Axis ◽  
Plasma Levels ◽  
Molecular Mechanisms ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Wky Rats ◽  
Glucocorticoid Signaling

So-ochim-tang-gamibang (SOCG) is a Korean traditional medicine; it has previously been shown to be safe and effective against depression. Persistently increased levels of circulating glucocorticoids have been considered as a pathological mechanism for depression and associated with decreased neurotrophic factors in the hippocampus. This study investigated whether SOCG controls the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and the molecular mechanisms underlying its effects in vivo and in vitro. Wistar Kyoto (WKY) rats were subjected to restraint stress, where SOCG was orally administered to the animals for 2 weeks. An open field test (OFT), forced swimming test (FST), and sucrose preference test (SPT) were performed to explore the antidepressant activity of SOCG in WKY rats. Plasma levels of HPA axis hormones were measured by ELISA or western blotting analysis. The expression levels or activation of HPA axis-related signaling molecules such as brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), extracellular regulated kinase (ERK), and glucocorticoid receptors (GRs) in the brain were determined by real-time PCR and western blotting analysis. Furthermore, a corticosterone- (CORT-) induced cell injury model was established using SH-SY5Y cells to explore the antidepressive effects of SOCG in vitro. The results of the OFT, FST, and SPT revealed that SOCG ameliorated depressive-like behaviors in the WKY rats. The blood plasma levels of HPA axis hormones such as CORT, CORT-releasing hormone (CRH), and adrenocorticotrophic hormone were downregulated by SOCG. On the other hand, SOCG upregulated the phosphorylation of CREB and ERK in both the rat hippocampus and CORT-treated SH-SY5Y cells. Moreover, it also increased the GR expression. These results suggested that SOCG may improve depression by controlling hyperactive glucocorticoid signaling via the downregulation of HPA axis hormones and upregulation of GR.

scholarly journals Effects of topical mechanical stability on the formation of Masquelet membrane in a rabbit radial defect model

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jie Xie ◽  
Donghao Liu ◽  
Haoyi Wang ◽  
Haitao Long ◽  
Yong Zhu ◽  
...  
Keyword(s):  
Western Blotting ◽  
Mechanical Stability ◽  
Mechanical Test ◽  
Control Group ◽  
Rigid Fixation ◽  
X Ray ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Fixation Group ◽  
Masquelet Technique

Abstract The exact mechanism of Masquelet technique is unknown. This study intends to explore the effects of topical mechanical stability on the formation of Masquelet membrane. Segmental radius shaft defect was created in all rabbits, which were filled with polymethylmethacrylate (PMMA) in Non-fixation group, and with PMMA fixed with plates in Fixation group, and subjected to no disposal in control group. The topical stability of PMMA and plates were monitored via X-ray and mechanical test. And the membranes were excised for further Histological, IHC and Western-Blotting analysis 4 and 6 weeks post-operatively. X-ray revealed no sign of plates loosening, or shift of PMMA. Mechanical tests revealed superior topical stability by plates. Pathological examinations suggested that vascularized and osteogenic membranes were formed around PMMA. IHC and Western-Blotting analysis revealed that both Fixation and Non-fixation group exerted significant effects on the expression of Ki67, COL I, and CD31 positive cells, as well as the protein expression of osteogenic (RUNX2, ALP) and angiogenic (VEGFA, TGF-β1) factors. And compared with membrane in Non-fixation group, Fixing PMMA spacer with plates caused a significant increase in osteogenic and angiogenic expression. This study indicates that rigid fixation provided by plate in Masquelet technique positively alters the quality of membrane formed surrounding PMMA, in terms of significantly osteogenic and angiogenic potential.

scholarly journals Western blotting analysis of heat shock proteins ofRickettsialesand other eubacteria

1998 ◽  
Vol 167 (2) ◽  
pp. 229-237 ◽  
Author(s):  
Marina E Eremeeva ◽  
Wei-Mei Ching ◽  
Yalin Wu ◽  
David J Silverman ◽  
Gregory A Dasch
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Western Blotting ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Shock Proteins

Some effects of a low sodium intake on the expression of P450 aldosterone synthase in the hamster adrenal cortex: immunoblotting, immunofluorescent and immuno-gold electron microscopic studies

1996 ◽  
Vol 149 (2) ◽  
pp. 341-349 ◽  
Author(s):  
J G LeHoux ◽  
A Lefebvre ◽  
L Ducharme ◽  
J Lehoux ◽  
D Martel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adrenal Cortex ◽  
Western Blotting ◽  
Sodium Intake ◽  
Electron Microscopic ◽  
Zonal Distribution ◽  
Aldosterone Synthase ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Low Sodium

Abstract In the current work we studied the effects of a low sodium intake on P450 aldosterone synthase (P450aldo) in the adrenal cortex of male hamsters by Western blotting analysis. We also investigated the zonal distribution of P450aldo with a specific antibody using immunofluorescence and immuno-gold electron microscopy. Western blotting analysis revealed a progressive induction of P450aldo in the adrenals of hamsters kept on a low sodium diet, with two-, four- and eightfold increases after 2, 4 and 21 days on the diet. Immunofluorescence microscopy showed that P450aldo was confined to the zona glomerulosa (ZG) cells. Electron microscopy showed P450aldo to be located in the mitochondria of ZG cells. When hamsters were maintained on a low sodium intake for 2, 11 and 21 days, P450aldo was still found only in the ZG; the ZG appeared either unchanged or sometimes slightly enlarged. Moreover, at days 11 and 21, the intensity of the immunofluorescent signal was much stronger in the ZG of hamsters on the low sodium intake than in controls. Hence, immunocytochemistry using the colloidal-gold technique showed P450aldo to be more abundant in the mitochondria of the experimental animals than in controls. To conclude, P450aldo is present only in the ZG of hamster adrenals and sodium restriction appears to induce its expression by stimulating production within individual ZG cells rather than by stimulating a proliferation of the ZG cells. Journal of Endocrinology (1996) 149, 341–349

The Expression of Human Leukocyte Formin Protein, a New Protein That Associates with AKT, Is Correlated with the Expression of ZAP-70 in CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1918-1918
Author(s):  
Patricia M.B. Favaro ◽  
Samuel S. Medina ◽  
Fabiola Traina ◽  
Gislaine B. Oliveira ◽  
Irene L. Metze ◽  
...  
Keyword(s):  
Prognostic Marker ◽  
Western Blotting ◽  
Expression Vector ◽  
Human Leukocyte ◽  
Control Group ◽  
Rapid Progression ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Spearman Test ◽  
New Protein

Abstract Recently, we have cloned a new human gene (GenBank Accession No. AY278319), belonging to the formin family. This new gene, called for us human leukocyte formin, presents the common domains found in formin-related proteins: FH1, FH2 and FH3 domains. Western Blotting analysis has demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies and cancer cell lines. Based on this pattern of expression, our objective was to investigate the expression of human leukocyte formin protein, by Western blotting analysis, in mononuclear cells from chronic lymphocytic leukemia (CLL) patients, isolated on a Ficoll-Hypaque gradient. We studied 18 CLL patients with median age of 65 y.o. (range, 45 to 86) out of treatment for at least three months (Rai 0 n=8; Rai 1 n=6; Rai 2 n=1; Rai3 n=1; Rai 4 n=2). As normal control we used 6 blood donors. Our data showed an overexpression of the human leukocyte formin in the CLL group when compared with the control group (p= 0.0354), as well as a positive correlation of this protein and ZAP-70 in the CLL group (Spearman test, p= 0.0107). The expression of ZAP-70 has been associated with rapid progression and poor survival and can be used as a prognostic marker. Previously we had described that human leukocyte formin protein associates with Akt, a critical survival regulator in many different cell types. The association was observed in a protein extract of Jurkat cell line and in peripheral blood leukocytes from CLL patients. In an attempt to confirm the association between Akt and human leukocyte formin, we performed cotransfections in 293 cells using an expression vector pEGFP containing FH2 or FH3 domains, and an expression vector of pCMV-HA containing the full length of Akt. The results showed that both FH2 and FH3 domains are involved in the association with Akt. The correlation of human leukocyte formin and ZAP-70 expression, and the association of human leukocyte formin protein with Akt suggest that this new protein may be involved in the signaling pathway of leukemia cell survival and is possibly a new prognostic marker.

Factor VIII A2 Domain Contains a Binding Site for Factor X

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4226-4226
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Midori Shima
Keyword(s):  
Binding Site ◽  
Western Blotting ◽  
Hemophilia A ◽  
Affinity Constant ◽  
Severe Bleeding ◽  
Dependent Manner ◽  
Severe Hemophilia ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
A2 Domain

Abstract Acquired hemophilia A (AHA) is a rare hemorrhagic disease in which autoantibodies against coagulation factor (F) VIII impair the coagulation system. The inhibitors developed in AHA are polyclonal autoantibodies and the majority of FVIII inhibitors bind to the A2, A3, or C2 domains. Depending on the location of the epitope, different mechanisms of action for the anti-FVIII antibodies have been reported. Anti-A3 antibodies neutralize the procoagulant activity of FVIII by preventing its interaction with FIXa. Anti-C2 antibodies inhibit the binding of FVIII to phospholipid membrane and/or von Willebrand factor, whereas A2 and A3 inhibitors block the binding of FVIII to FIXa and FX, respectively, and obstruct the formation of the Xase complex. We have a case of AHA whose inhibitor recognizes only A2 domain and attempted several approaches to determine the mechanism of neutralizing FVIII. Thrombin and plasmin generation assay using patient’s plasma showed that the thrombin and plasmin generation in this AHA patient were decreased compared with that in congenital severe hemophilia A patient. Furthermore, FX generation (Coatest) in this AHA was also decreased compared with that in congenital severe hemophilia A patient (p<0.05). These results indicated that this inhibitor impaired the generation of Xase complex and might cause the severe bleeding disorder in this patient. The IgG subclass of inhibitor in our case was IgG1 and IgG4. Western blotting analysis using FVIIIa revealed that the inhibitor IgG recognized only A2 domain. Furthermore, western blotting analysis using FVIII A2 fragment, digested by activated protein C, showed that the inhibitor IgG bound to FVIII A2N (residue 372-562) fragment. It is known that FVIII A2 domain contains FIXa and thrombin binding sites. Western blotting analysis revealed that the inhibitor IgG inhibited Arg336 cleavage in FVIIIa by FIXa and Arg372 cleavage in FVIII by thrombin. However, the FXa-catalyzed cleavage at Arg372 in FVIII was inhibited by this inhibitor IgG. ELISA-based assay showed that the inhibitor IgG inhibited FX binding to FVIII A2. These results suggest that FX(a) binds to FVIII A2 domain. Therefore, to determine the direct binding of FX and FVIII A2 domain, ELISA-based assay was employed to assess this interaction. ELISA-based assay showed that FVIII A2 fragment bound FX in a dose-dependent manner with moderate affinity (Kd = 338 nM). FX inhibited FVIII A2 fragment binding to immobilized FX up to 70% with an inhibition constant (Ki = 254 nM) similar to the affinity constant. It is known that the residue 484-509 in the A2 domain interacts with FIXa. We hypothesized that FX binding site in the A2 domain might be in the opposite side of FIXa binding site in the A2 domain. According to the 3-D model of FVIII molecule, we prepared synthetic peptides corresponding to FVIII A2 residues 400-409, 409-419, and 420-429. To determine the specificity of these sequences for FX interaction, we examined the effects of these peptides on FVIII A2 binding to FX using ELISA-based assay. The 400-409 peptide inhibited the A2 and FX interaction up to 70%. In contrast, the 410-419 and the 420-429 peptides inhibited the interaction up to 30%. Covalent cross-linking was observed between the 400-409 peptide and FX following reaction with EDC using SDS-PAGE. These results indicate that FVIII A2 domain contains the binding site for FX(a), and the 400-409 region in the FVIII A2 domain contributes to a unique FX(a)-interactive site. Disclosures No relevant conflicts of interest to declare.

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