| 1. |
- Qi, Xiaoying, et al.
(författare)
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High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)
- 2018
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :138
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Tidskriftsartikel (refereegranskat)abstract
- Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.
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| 2. |
- Zhang, Xiaoying, et al.
(författare)
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IL-6 Regulates MMP-10 Expression via JAK2/STAT3 Signaling Pathway in a Human Lung Adenocarcinoma Cell Line
- 2009
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Ingår i: Anticancer research. - 1791-7530. ; 29:11, s. 4497-4501
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Tidskriftsartikel (refereegranskat)abstract
- We previously reported that matrix metalloproteinase (MMP)-10 mRNA levels were significantly lower in tumor tissues than in adjacent normal tissues in human non-small cell lung cancer (NSCLC), whereas protein levels of MMP-10 were higher in the tumor tissues than the adjacent tissues. The mechanism of this divergence is still unknown. In the present stud), the role of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) on interleukin (IL)-6 mediated regulation of MMP-10 expression was investigated in a human lung adenocarcinoma cell line (A549 cells) and the molecular regulatory mechanism of MMP-10 expression was explored. A549 cells were stimulated by different concentrations of IL-6 with or without AG490, a specific JAK2 inhibitor. It was demonstrated that IL-6 moderately reduced the MMP-10 mRNA levels, whereas it significantly enhanced the MMP-10 protein mass in the A549 cells. This phenomenon mimicked the divergence of mRNA level and protein mass of MMP-10 in human NSCLC. Moreover, the present study indicated that IL-6 regulation of MMP-10 expression was via the JAK2/STAT3 pathway. STAT3 mRNA levels were significantly increased when the cells were treated with IL-6, whereas when AG490 (50 mu M) was added to the cell cultures, IL-6-induced increase of STAT3 mRNA levels was abolished. Meanwhile, AG490 blocked the IL-6-induced inhibition of MMP-10 mRNA as well as blocking the IL-6-induced increase of MMP-10 protein mass in the A549 cells. Neither IL-6 nor AG490 influenced JAK2 mRNA levels in the A549 cell cultures. It is concluded that the JAK2/STAT3 pathway is involved in the IL-6-mediated regulation of MMP-10, and IL-6 can moderately reduce MMP-10 mRNA levels and strongly increase MMP-10 protein mass in human lung adenocarcinoma A549 cells. Contrasting effects of IL-6 on MMP-10 mRNA level and protein concentration in A549 cells may partially explain the divergence of MMP-10 mRNA level and protein mass in human NSCLC.
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| 3. |
- Zhu, Yanping, et al.
(författare)
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Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB)
- 2019
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Ingår i: Clinical Proteomics. - : BMC. - 1542-6416 .- 1559-0275. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice.Methods: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry.Results: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC.Conclusion: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.
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| 4. |
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| 5. |
- Luo, Guanghua, et al.
(författare)
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Genotyping of single nucleotide polymorphisms using base-quenched probe: A method does not invariably depend on the deoxyguanosine nucleotide
- 2009
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 386:2, s. 161-166
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Tidskriftsartikel (refereegranskat)abstract
- Most available methods for detecting single nucleotide polymorphisms (SNPs) are based principally on the system that can produce an increased fluorescence signal during hybridization. In the current study, we demonstrate a method of base-quenched probe for polymerase chain reaction (PCR) genotyping that requires only a pair of primers and one fluorescent probe and does not invariably depend on the deoxyguanosine nucleotide. This method further exploits the phenomenon of fluorescence quenching of fluorescent-labeled probe during hybridization to its complementary target gene's sequence. 6-Carboxyfluorescein (FAM) can be directly conjugated to a base of either adenine (A), thymine (T), cytosine (C), or guanine (G), referred to as A-, T-, C-, or G-quenched probe, respectively, at either the 5' or 3' end. For describing the method in detail, we chose apolipoprotein M (apoM) as a target gene in the current study. DNA sequencing analyses validated that all four types of base-quenched probes could provide unbiased genotyping results (K = 1, P = 0.000), although the maximum speed of fluorescence increase, max(dF/dT), when using the G-quenched probe method, was approximately twofold lower than the others (P < 0.0001). Moreover, we applied this method to detect another seven SNPs in the genomes of phospholipase A2, monocyte chemoattractant protein I (MCP1), and L-ficolin, further confirming our method. It is concluded that this method is precise, simple, and economic as well as suitable for large-scale genotyping Studies. (C) 2008 Elsevier Inc. All rights reserved.
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| 6. |
- Luo, Guanghua, et al.
(författare)
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Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPARβ/δ in HepG2 cells.
- 2014
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 445:1, s. 203-207
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Tidskriftsartikel (refereegranskat)abstract
- It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPARβ/δ pathway.
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| 7. |
- Luo, Guanghua, et al.
(författare)
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Rosiglitazone Enhances Apolipoprotein M (Apom) Expression in Rat's Liver.
- 2014
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Ingår i: International Journal of Medical Sciences. - : Ivyspring International Publisher. - 1449-1907. ; 11:10, s. 1015-1021
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (APOM) has been suggested as a vasculoprotective constituent of high density lipoprotein (HDL), which plays a crucial role behind the mechanism of HDL-mediated anti-atherosclerosis. Previous studies demonstrated that insulin resistance could associate with decreased APOM expressions. In agreement with our previous reports, here, we further confirmed that the insulin sensitivity was also reduced in rats treated with high concentrations of glucose; such effect could be reversed by administration of rosiglitazone, a peroxisome proliferator-activated receptor-γ (PPARγ). The present study shows that Apom expression is significantly affected by either rosiglitazone or hyperglycemia alone without cross interaction with each other, which indicates that the pathway of Apom expression regulating by hyperglycemia might be differed from that by rosiglitazone. Further study indicated that hyperglycemia could significantly inhibit mRNA levels of Lxrb (P=0.0002), small heterodimer partner 1 (Shp1) (P<0.0001), liver receptor homologue-1 (Lrh1) (P=0.0012), ATP-binding cassette transporter 1 (Abca1) (P=0.0012) and Pparb/d (P=0.0043). Two-way ANOVA analysis demonstrated that the interactions between rosiglitazone and infusion of 25% glucose solution on Shp1 (P=0.0054) and Abca1 (4E, P=0.0004) mRNA expression was statistically significant. It is concluded that rosiglitazone could increase Apom expression, of which the detailed mechanism needs to be further investigated. The downregulation of Apom by hyperglycemia might be mainly through decreasing expression of Pparg and followed by inhibiting Lxrb in rats.
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| 8. |
- Shi, Yuanping, et al.
(författare)
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Comprehensive lipidomics in apoM−/− mice reveals an overall state of metabolic distress and attenuated hepatic lipid secretion into the circulation
- 2020
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Ingår i: Journal of Genetics and Genomics. - : Elsevier BV. - 1673-8527. ; 47:9, s. 523-534
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (apoM) participates in both high-density lipoprotein and cholesterol metabolism. Little is known about how apoM affects lipid composition of the liver and serum. In this study, we systemically investigated the effects of apoM on liver and plasma lipidomes and how apoM participates in lipid cycling, via apoM knockout in mice and the human SMMC-7721 cell line. We used integrated mass spectrometry–based lipidomics approaches to semiquantify more than 600 lipid species from various lipid classes, which include free fatty acids, glycerolipids, phospholipids, sphingolipids, glycosphingolipids, cholesterol, and cholesteryl esters (CEs), in apoM−/− mouse. Hepatic accumulation of neutral lipids, including CEs, triacylglycerols, and diacylglycerols, was observed in apoM−/− mice; while serum lipidomic analyses showed that, in contrast to the liver, the overall levels of CEs and saturated/monounsaturated fatty acids were markedly diminished. Furthermore, the level of ApoB-100 was dramatically increased in the liver, whereas significant reductions in both ApoB-100 and low-density lipoprotein (LDL) cholesterol were observed in the serum of apoM−/− mice, which indicated attenuated hepatic LDL secretion into the circulation. Lipid profiles and proinflammatory cytokine levels indicated that apoM−/− leads to hepatic steatosis and an overall state of metabolic distress. Taken together, these results revealed that apoM knockout leads to hepatic steatosis, impaired lipid secretion, and an overall state of metabolic distress.
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| 9. |
- Shi, Yuanping, et al.
(författare)
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Increased expression levels of inflammatory cytokines and adhesion molecules in lipopolysaccharide‑induced acute inflammatory apoM‑/‑ mice
- 2020
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Ingår i: Molecular Medicine Reports. - : Spandidos Publications. - 1791-2997 .- 1791-3004. ; 22:4, s. 3117-3126
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Tidskriftsartikel (refereegranskat)abstract
- apolipoproteinM(apoM)mayserveaprotectiverole inthedevelopmentofinflammation.Nuclearfactor‑κB(nF-κB) and its downstream factors (including a number of inflammatory cytokines and adhesion molecules) are essential for the regulation of inflammatory processes. In the present study, the importance of apoM in lipopolysaccharide (LPS)‑induced acute inflammation and its potential underlying mechanisms, were investigated using an apoM‑knockout mouse model. The levels of inducible nitric oxide synthase (iNOS), NF‑κB, interleukin (IL)‑1β, intercellular adhesion molecule 1 (ICAM‑1) and vascular cell adhesion protein 1 (VCAM‑1) were detected using reverse transcription‑quantitative PCR and western blotting. The serum levels of IL‑6 and IL‑10 were detected using Luminex technology. The results demonstrated that the protein levels of inoS, nF-κB, il-1β, ICAM‑1 and VCAM‑1 were significantly increased in apoM-/- mice compared with those in apoM+/+ mice. In addition, two‑way ANOVA revealed that the interaction between apoM and LPS had a statistically significant effect on a number of factors, including the mRNA expression levels of hepatic iNOS, NF‑κB, il-1β, icaM-1 and VCAM‑1. Notably, the effects of apoM and 10 mg/kg LPS on the levels of IL‑6 and IL‑10 were the opposite of those induced by 5 mg/kg LPS, which could be associated with the dual anti‑ and pro‑inflammatory effects of IL‑6 and IL‑10. Collectively, the results of the present study revealed that apoM is an important regulator of inflammatory cytokine and adhesion molecule production in LPS‑induced inflammation, which may consequently be associated with the severity of inflammation. These findings indicated that the anti‑inflammatory effects of apoM may partly result from the inhibition of the nF-κB pathway.
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| 10. |
- Swami, Viren, et al.
(författare)
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The Attractive Female Body Weight and Female Body Dissatisfaction in 26 Countries Across 10 World Regions : Results of the International Body Project I
- 2010
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Ingår i: Personality and Social Psychology Bulletin. - : Sage Publications. - 0146-1672 .- 1552-7433. ; 36:3, s. 309-325
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Tidskriftsartikel (refereegranskat)abstract
- This study reports results from the first International Body Project (IBP-I), which surveyed 7,434 individuals in 10 major world regions about body weight ideals and body dissatisfaction. Participants completed the female Contour Drawing Figure Rating Scale (CDFRS) and self-reported their exposure to Western and local media. Results indicated there were significant cross-regional differences in the ideal female figure and body dissatisfaction, but effect sizes were small across high-socioeconomic-status (SES) sites. Within cultures, heavier bodies were preferred in low-SES sites compared to high-SES sites in Malaysia and South Africa (ds = 1.94-2.49) but not in Austria. Participant age, body mass index (BMI), and Western media exposure predicted body weight ideals. BMI and Western media exposure predicted body dissatisfaction among women. Our results show that body dissatisfaction and desire for thinness is commonplace in high-SES settings across world regions, highlighting the need for international attention to this problem.
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