45 research outputs found

    APN secreted by PVAT aggravates autophagy in plaque.

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    <p>A, Representative western blot of LC3 expression in arteria carotis transplanted with WT or APN<sup>-/-</sup> PVAT 4 weeks after atherosclerosis (n = 6 per group). B, Quantitative analysis of LC3 protein expression in various groups. *P<0.05 versus (WT) PVAT. C, Immunofluorescence of p62, another marker of autophagy, in the arteria carotis with WT or APN<sup>-/-</sup> PVAT (n = 6 per group). D, Histogram shows p62 positive cells per 100 cells. *P<0.05 versus (WT) PVAT.</p

    APN induces autophagy in macrophage through Akt-FOXO3a pathway.

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    <p>A, Western blot shows the protein level of p-Akt, Akt, p-FOXO3a, FOXO3a in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. B, Western blot shows the protein level of PTEN, p-mTOR, mTOR in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. C, Quantitative analysis of p-Akt/Akt ratio, p-FOXO3a/FOXO3a ratio and p-mTOR/mTOR ratio in macrophages stimulated with phosphate buffered saline, APN, and APN in combination with 740Y-P, respectively. n = 6 per group. *P<0.05 versus macrophage treated with saline. D, Quantification of the optical density of PTEN in each groups. n = 6 per group. *P<0.05 versus macrophage treated with saline.</p

    APN exacerbates macrophage autophagy in vitro.

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    <p>A and B respectively show the representative western blot and quantitative analysis of LC3 protein level in VSMC and macrophage stimulated with or without APN (5 ÎĽg/ml). n = 6 per group. *P<0.05 versus macrophage without APN. C and D respectively show the western blot and quantitative analysis of P62 and Beclin 1 protein level in macrophage treated with or without APN (5 ÎĽg/ml). n = 6 per group. *P<0.05 versus macrophage without APN.</p

    Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification–Mass Spectrometry-based Protein–Protein Interaction Analysis

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    Affinity purification-mass spectrometry (AP–MS) has become the method of choice for discovering protein–protein interactions (PPIs) under native conditions. The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs. Here, we used NFκB/RelA and Bromodomain-containing protein 4 (BRD4) as baits and test five distinct trypsin digestion methods (two using “on-beads,” three using “elution-digestion” protocols). Although the performance of the trypsin digestion protocols change slightly depending on the different baits, antibodies and cell lines used, we found that elution-digestion methods consistently outperformed on-beads digestion methods. The high-abundance interactors can be identified universally by all five methods, but the identification of low-abundance RelA interactors is significantly affected by the choice of trypsin digestion method. We also found that different digestion protocols influence the selected reaction monitoring (SRM)–MS quantification of PPIs, suggesting that optimization of trypsin digestion conditions may be required for robust targeted analysis of PPIs

    Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples

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    Protein tyrosine nitration by oxidative and nitrate stress is important in the pathogenesis of many inflammatory or aging-related diseases. Mass spectrometry analysis of protein nitrotyrosine is very challenging because the non-nitrated peptides suppress the signals of the low-abundance nitrotyrosine (NT) peptides. No validated methods for enrichment of NT-peptides are currently available. Here we report an immunoaffinity enrichment of NT-peptides for proteomics analysis. The effectiveness of this approach was evaluated using nitrated protein standards and whole-cell lysates in vitro. A total of 1881 NT sites were identified from a nitrated whole-cell extract, indicating that this immunoaffinity-MS method is a valid approach for the enrichment of NT-peptides, and provides a significant advance for characterizing the nitrotyrosine proteome. We noted that this method had higher affinity to peptides with N-terminal nitrotyrosine relative to peptides with other nitrotyrosine locations, which raises the need for future study to develop a pan-specific nitrotyrosine antibody for unbiased, proteome-wide analysis of tyrosine nitration. We applied this method to quantify the changes in protein tyrosine nitration in mouse lungs after intranasal poly­(I:C) treatment and quantified 237 NT sites. This result indicates that the immunoaffinity-MS method can be used for quantitative analysis of protein nitrotyrosines in complex samples

    Comparison of the proteins in one published human glomerular proteome dataset with the renal cortical proteome listed in Additional file

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    . the diagram of the overlapping between the two datasets. . Subcellular location distribution patterns of the 100 most abundant proteins in two datasets.<p><b>Copyright information:</b></p><p>Taken from "Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry"</p><p>http://www.proteomesci.com/content/6/1/15</p><p>Proteome Science 2008;6():15-15.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2412861.</p><p></p

    NSAF values for every protein see Additional file are shown graphically

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    NSAF values of identified proteins fall within the range from 1.7 × 10to 1.8 × 10.<p><b>Copyright information:</b></p><p>Taken from "Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry"</p><p>http://www.proteomesci.com/content/6/1/15</p><p>Proteome Science 2008;6():15-15.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2412861.</p><p></p

    IPA classification of all identified proteins by subcellular location and protein families

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    <p><b>Copyright information:</b></p><p>Taken from "Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry"</p><p>http://www.proteomesci.com/content/6/1/15</p><p>Proteome Science 2008;6():15-15.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2412861.</p><p></p

    Characteristics of included studies.

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    <p>PCI = percutaneous coronary intervention; CK-MB = creatine kinase-MB; UNL = upper normal limit of normal; NSTE-ACS = non-ST-segment elevation acute coronary syndrome;</p><p>Characteristics of included studies.</p

    Characteristics of included studies.

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    <p>PCI = percutaneous coronary intervention; CK-MB = creatine kinase-MB; UNL = upper normal limit of normal; NSTE-ACS = non-ST-segment elevation acute coronary syndrome; ROMA trial = Rosuvastatin pretreatment in patients undergoing elective PCI to reduce the incidence of periprocedural myocardial necrosis; ROMA II trial = Comparison of high reloading Rosuvastatin and Atorvastatin pretreatment in patients undergoing elective PCI to reduce the incidence of periprocedural myocardial necrosis; NA = not available.</p><p>Characteristics of included studies.</p
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