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    Agilent technologies two dimensional liquid chromatography mass spectrometry
    Two Dimensional Liquid Chromatography Mass Spectrometry, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reversed-phase Chromatography:

    Article Title: Proteomic analysis of human periodontal ligament cells under hypoxia
    Article Snippet: .. Two-dimensional liquid chromatography-mass spectrometry (2D-LC-MSMS) analysis including reversed-phase chromatographic separation (Agilent Technologies, Santa Clara, CA, USA) and reversed-phase chromatography on a TripleTOF (AB SCIEX, USA) was conducted. ..

    Chromatography:

    Article Title: Proteomic analysis of human periodontal ligament cells under hypoxia
    Article Snippet: .. Two-dimensional liquid chromatography-mass spectrometry (2D-LC-MSMS) analysis including reversed-phase chromatographic separation (Agilent Technologies, Santa Clara, CA, USA) and reversed-phase chromatography on a TripleTOF (AB SCIEX, USA) was conducted. ..

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    Agilent technologies mast1 variants
    CHIP ubiquitinates and degrades <t>MAST1</t> when unmasked by hsp90B. ( A ) MAST1 interacts with CHIP in cells. GST-pull-down samples from 293T were applied to LC-MS/MS. Spectral counts of CHIP and MAST1 in samples treated with or without 17-AAG are shown. ( B ) Overexpressed CHIP and MAST1 interact in cancer cells. Effect of CHIP overexpression ( C ) or knockout ( D ) on MAST1 levels. ( E ) Comparison of MAST1 protein stability in cells with CHIP modulation was achieved by cycloheximide (CHX) chase assay. Cells with CHIP knockdown were transfected with shRNA-resistant CHIP variants followed by 5 μg/mL CHX treatment for the indicated time. MAST1 amount was determined by density analysis. Representative data are shown. ( F ) In vitro CHIP ubiquitination assay using purified MAST1 WT or 2KR. ( G ) Ubiquitination of MAST1 WT and 2KR in cells. GST pull-down samples from A549 cisR cells treated with MG-132 (10 μM) were immunoblotted with anti-ubiquitin antibody. ( H ) Interaction of MAST1 WT or 2KR, hsp90B, and CHIP in the presence or absence of 17-AAG. MAST1 WT and 2KR protein stability in the absence ( I ) and presence ( J ) of 17-AAG was determined by cycloheximide chase assay. Data shown are representative of 3 ( B – E , I , and J ) and 2 ( F , G , H ) independent biological experiments. Data are mean ± SD from 3 technical replicates. Statistical analysis was performed by 2-way ANOVA for E and 1-way ANOVA for I and J . *** P
    Mast1 Variants, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies plasma pge2
    Baseline plasma <t>PGE2</t> is elevated in NYUHJD learning, NYUHJD progression and Pfizer cohorts. Plasma levels of PGE2 were determined using ACE EIA kit as described in Methods. The NYUHJD learning cohort (A) consists of symptomatic knee osteoarthritis (SKOA)
    Plasma Pge2, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies q tof lc esi ms ms instrument
    Proteins differentially expressed by HTR-8/SVneo cells cultured under serum-depleted conditions. a HTR-8/SVneo cells were grown in medium with 10 or 0 % FBS for 24 h (in triplicate). Total protein extracts (0.50 mg) were separated by 18 cm 2DE gels as previously described ( n = 3). The protein spots were analyzed via LC <t>ESI-Q/TOF</t> and identified using bioinformatics analysis. MW – molecular weight marker (RPN5800, GE). b Effect of serum depletion on the expression of some trophoblast proteins. Statistical analysis of 2DE protein spots identified by MS/MS in HTR-8/SVneo proteomes either with 10 % FBS or without FBS ( n = 3). Kolmogorov-Smirnov test (D value): 0.10, D > 0.642 (a, D = 1.0; b, D = 0.67)
    Q Tof Lc Esi Ms Ms Instrument, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies two dimensional lc maldi tof tof analysis
    Isobaric tags for relative quantification (iTRAQ) of modulated proteins in 3T3L1-PG cells. a Workflow followed for the relative quantification of modulated proteins in PG-3T3L1 cells. b Summary of the protein identification and relative quantification. c Significantly modulated biological processes after String 9.0 analysis of the modulated individual proteins. GFP green fluorescent protein, GO gene ontology, <t>LC-MALDI-TOF</t> liquid chromatography coupled offline to matrix-assisted laser desorption ionization–time of flight, PG progerin
    Two Dimensional Lc Maldi Tof Tof Analysis, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CHIP ubiquitinates and degrades MAST1 when unmasked by hsp90B. ( A ) MAST1 interacts with CHIP in cells. GST-pull-down samples from 293T were applied to LC-MS/MS. Spectral counts of CHIP and MAST1 in samples treated with or without 17-AAG are shown. ( B ) Overexpressed CHIP and MAST1 interact in cancer cells. Effect of CHIP overexpression ( C ) or knockout ( D ) on MAST1 levels. ( E ) Comparison of MAST1 protein stability in cells with CHIP modulation was achieved by cycloheximide (CHX) chase assay. Cells with CHIP knockdown were transfected with shRNA-resistant CHIP variants followed by 5 μg/mL CHX treatment for the indicated time. MAST1 amount was determined by density analysis. Representative data are shown. ( F ) In vitro CHIP ubiquitination assay using purified MAST1 WT or 2KR. ( G ) Ubiquitination of MAST1 WT and 2KR in cells. GST pull-down samples from A549 cisR cells treated with MG-132 (10 μM) were immunoblotted with anti-ubiquitin antibody. ( H ) Interaction of MAST1 WT or 2KR, hsp90B, and CHIP in the presence or absence of 17-AAG. MAST1 WT and 2KR protein stability in the absence ( I ) and presence ( J ) of 17-AAG was determined by cycloheximide chase assay. Data shown are representative of 3 ( B – E , I , and J ) and 2 ( F , G , H ) independent biological experiments. Data are mean ± SD from 3 technical replicates. Statistical analysis was performed by 2-way ANOVA for E and 1-way ANOVA for I and J . *** P

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: CHIP ubiquitinates and degrades MAST1 when unmasked by hsp90B. ( A ) MAST1 interacts with CHIP in cells. GST-pull-down samples from 293T were applied to LC-MS/MS. Spectral counts of CHIP and MAST1 in samples treated with or without 17-AAG are shown. ( B ) Overexpressed CHIP and MAST1 interact in cancer cells. Effect of CHIP overexpression ( C ) or knockout ( D ) on MAST1 levels. ( E ) Comparison of MAST1 protein stability in cells with CHIP modulation was achieved by cycloheximide (CHX) chase assay. Cells with CHIP knockdown were transfected with shRNA-resistant CHIP variants followed by 5 μg/mL CHX treatment for the indicated time. MAST1 amount was determined by density analysis. Representative data are shown. ( F ) In vitro CHIP ubiquitination assay using purified MAST1 WT or 2KR. ( G ) Ubiquitination of MAST1 WT and 2KR in cells. GST pull-down samples from A549 cisR cells treated with MG-132 (10 μM) were immunoblotted with anti-ubiquitin antibody. ( H ) Interaction of MAST1 WT or 2KR, hsp90B, and CHIP in the presence or absence of 17-AAG. MAST1 WT and 2KR protein stability in the absence ( I ) and presence ( J ) of 17-AAG was determined by cycloheximide chase assay. Data shown are representative of 3 ( B – E , I , and J ) and 2 ( F , G , H ) independent biological experiments. Data are mean ± SD from 3 technical replicates. Statistical analysis was performed by 2-way ANOVA for E and 1-way ANOVA for I and J . *** P

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Chromatin Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Over Expression, Knock-Out, Transfection, shRNA, In Vitro, Ubiquitin Assay, Purification

    Combination of 17-AAG and lestaurtinib further inhibits MAST1 activity and cisplatin-resistant tumor growth. ( A and B ) Combination effect of 17-AAG and lestaurtinib on expression and activity of MAST1. KB-3-1 cisR and A549 cisR cells were treated with 17-AAG (100 nM) and lestaurtinib (100 nM) in the presence of sublethal doses of cisplatin for 24 hours. The kinase activity of MAST1 was assessed by phospho-MEK1 S217/S221 level ( A ) and ADP-Glo Kinase assay ( B ). Effect of combinatorial treatment with 17-AAG and lestaurtinib on cell viability ( C ) and cisplatin sensitivity ( D ). ( E ) Combination index (CI) plots are shown for diverse cisplatin-resistant cancer cell lines. ( F – H ) Effect of cisplatin treatment with the combination of 17-AAG and lestaurtinib on tumor growth of lung cancer PDX mice. Mice were administered 17-AAG, lestaurtinib, and cisplatin from 28 days after xenograft. Tumor volume ( F ) and tumor weight ( G ) are shown. ( H and I ) The kinase activity of MAST1 in PDX tumors. Data shown are representative of 3 ( A , C – E , and H ) and 2 ( B and I ) independent biological experiments. Data are mean ± SD from 3 technical replicates for B – D and I . Error bars indicate SEM ( F ) and SD ( G ) in 5 mice/group. Statistical analysis was performed by 2-way ANOVA for F and 1-way ANOVA for all the rest. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: Combination of 17-AAG and lestaurtinib further inhibits MAST1 activity and cisplatin-resistant tumor growth. ( A and B ) Combination effect of 17-AAG and lestaurtinib on expression and activity of MAST1. KB-3-1 cisR and A549 cisR cells were treated with 17-AAG (100 nM) and lestaurtinib (100 nM) in the presence of sublethal doses of cisplatin for 24 hours. The kinase activity of MAST1 was assessed by phospho-MEK1 S217/S221 level ( A ) and ADP-Glo Kinase assay ( B ). Effect of combinatorial treatment with 17-AAG and lestaurtinib on cell viability ( C ) and cisplatin sensitivity ( D ). ( E ) Combination index (CI) plots are shown for diverse cisplatin-resistant cancer cell lines. ( F – H ) Effect of cisplatin treatment with the combination of 17-AAG and lestaurtinib on tumor growth of lung cancer PDX mice. Mice were administered 17-AAG, lestaurtinib, and cisplatin from 28 days after xenograft. Tumor volume ( F ) and tumor weight ( G ) are shown. ( H and I ) The kinase activity of MAST1 in PDX tumors. Data shown are representative of 3 ( A , C – E , and H ) and 2 ( B and I ) independent biological experiments. Data are mean ± SD from 3 technical replicates for B – D and I . Error bars indicate SEM ( F ) and SD ( G ) in 5 mice/group. Statistical analysis was performed by 2-way ANOVA for F and 1-way ANOVA for all the rest. * P

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Activity Assay, Expressing, Kinase Assay, Mouse Assay

    Hsp90B-MAST1-CHIP signaling correlates with cisplatin resistance in human head and neck cancer. ( A ) Tumor samples from HNSCC patients who received platinum-based chemotherapy. Representative IHC staining images of hsp90B, CHIP, and MAST1 for 0, +1, +2, and +3 scores are shown. Scale bars = 50 μm. The levels of hsp90B ( B ) and CHIP ( C ) were monitored by IHC in tumors from HNSCC patients who were sensitive or resistant to platinum-based (cisplatin or carboplatin) chemotherapy. The correlation matrix heatmaps between MAST1 and hsp90B ( D ) or CHIP ( E ) in HNSCC patients who received platinum-based chemotherapy. r represents Pearson’s correlation coefficient. For B and C , n = 30 (open circles; platinum-sensitive group) and n = 46 (closed gray circles; platinum-resistant group); for D and E , n = 76. P values were determined by 2-tailed Student’s t test for B and C , and χ 2 test for D and E .

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: Hsp90B-MAST1-CHIP signaling correlates with cisplatin resistance in human head and neck cancer. ( A ) Tumor samples from HNSCC patients who received platinum-based chemotherapy. Representative IHC staining images of hsp90B, CHIP, and MAST1 for 0, +1, +2, and +3 scores are shown. Scale bars = 50 μm. The levels of hsp90B ( B ) and CHIP ( C ) were monitored by IHC in tumors from HNSCC patients who were sensitive or resistant to platinum-based (cisplatin or carboplatin) chemotherapy. The correlation matrix heatmaps between MAST1 and hsp90B ( D ) or CHIP ( E ) in HNSCC patients who received platinum-based chemotherapy. r represents Pearson’s correlation coefficient. For B and C , n = 30 (open circles; platinum-sensitive group) and n = 46 (closed gray circles; platinum-resistant group); for D and E , n = 76. P values were determined by 2-tailed Student’s t test for B and C , and χ 2 test for D and E .

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Chromatin Immunoprecipitation, Immunohistochemistry, Staining

    Inhibition of hsp90B sensitizes cisplatin-resistant cancer cells to cisplatin through MAST1. ( A ) Cisplatin IC 50 upon 17-AAG treatment (50 nM, 48 hours) with or without MAST1 knockdown. Cisplatin IC 50 values were determined by CellTiter-Glo assay and analyzed by GraphPad Prism 8. ( B ) Effect of 17-AAG and MAST1 knockdown on tumor growth of cisplatin-treated xenograft mice. Mice were treated with cisplatin (5 mg/kg) and 17-AAG (50 mg/kg) from 5 days after xenograft. Tumor volume (left) and tumor weight (right) for each group and MAST1 expression in tumor lysates are shown. Cisplatin IC 50 ( C ) and cisplatin-resistant tumor growth ( D ) upon 17-AAG treatment, MAST1 knockdown, and rescue expression of MAST1 WT. Cell viability assay and xenograft assay were performed as in A and B . ( E ) Cell proliferation of KB-3-1 cisR and A549 cisR cells with hsp90B knockdown and MAST1 overexpression in the presence of cisplatin. Cells were treated with sublethal doses of cisplatin (5 μg/mL KB-3-1 cisR ; 2 μg/mL A549 cisR ) and proliferation was determined by trypan blue exclusion. ( F ) Effect of hsp90B knockdown and MAST1 overexpression on cisplatin-resistant tumor growth. Mice were treated with cisplatin (5 mg/kg) from 5 days after xenograft. Tumor volume (left) and tumor weight (right) for each group and hsp90B and MAST1 expression in tumor lysates are shown. Data shown are representative of 2 ( A – D and F ) and 3 ( E ) independent biological experiments. Data are mean ± SD from 3 technical replicates for A , C , and E ; n = 6 for B , D , and F . Error bars represent SEM for tumor volume and SD for tumor weight. Statistical analysis was performed by 2-way ANOVA for B , D , and F (left), and E , and 1-way ANOVA for A and C and B , D , and F (right). * P

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: Inhibition of hsp90B sensitizes cisplatin-resistant cancer cells to cisplatin through MAST1. ( A ) Cisplatin IC 50 upon 17-AAG treatment (50 nM, 48 hours) with or without MAST1 knockdown. Cisplatin IC 50 values were determined by CellTiter-Glo assay and analyzed by GraphPad Prism 8. ( B ) Effect of 17-AAG and MAST1 knockdown on tumor growth of cisplatin-treated xenograft mice. Mice were treated with cisplatin (5 mg/kg) and 17-AAG (50 mg/kg) from 5 days after xenograft. Tumor volume (left) and tumor weight (right) for each group and MAST1 expression in tumor lysates are shown. Cisplatin IC 50 ( C ) and cisplatin-resistant tumor growth ( D ) upon 17-AAG treatment, MAST1 knockdown, and rescue expression of MAST1 WT. Cell viability assay and xenograft assay were performed as in A and B . ( E ) Cell proliferation of KB-3-1 cisR and A549 cisR cells with hsp90B knockdown and MAST1 overexpression in the presence of cisplatin. Cells were treated with sublethal doses of cisplatin (5 μg/mL KB-3-1 cisR ; 2 μg/mL A549 cisR ) and proliferation was determined by trypan blue exclusion. ( F ) Effect of hsp90B knockdown and MAST1 overexpression on cisplatin-resistant tumor growth. Mice were treated with cisplatin (5 mg/kg) from 5 days after xenograft. Tumor volume (left) and tumor weight (right) for each group and hsp90B and MAST1 expression in tumor lysates are shown. Data shown are representative of 2 ( A – D and F ) and 3 ( E ) independent biological experiments. Data are mean ± SD from 3 technical replicates for A , C , and E ; n = 6 for B , D , and F . Error bars represent SEM for tumor volume and SD for tumor weight. Statistical analysis was performed by 2-way ANOVA for B , D , and F (left), and E , and 1-way ANOVA for A and C and B , D , and F (right). * P

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Inhibition, Glo Assay, Mouse Assay, Expressing, Viability Assay, Xenograft Assay, Over Expression

    Inhibition of hsp90 induces ubiquitination of MAST1 at lysine 317/545 that leads to proteasomal degradation. ( A ) Effect of 17-AAG on MAST1 ubiquitination. 293T cells with GST-MAST1 and HA-tagged ubiquitin (Ub) were treated with 17-AAG for 4 hours and subjected to GST pull down. Anti-HA antibody was used to detect ubiquitinated MAST1. ( B and C ) Effect of 17-AAG on MAST1 proteasomal degradation. Cells were treated with or without MG-132 (10 μM) before addition of 17-AAG (1 μM) in 293T ( B ) or cisplatin-resistant cancer cells ( C ), and exogenous or endogenous MAST1 levels were detected, respectively. cRaf and AKT levels are shown for comparison. ( D ) Effect of 17-AAG on MAST1 ubiquitination and proteasomal degradation in cisplatin-resistant cancer cells. ( E ) MS spectra of ubiquitinated peptide fragments of MAST1. 293T cells with GST-MAST1 were treated with 1 μM of 17-AAG for 4 hours. Ubiquitination at K317 and K545 in MAST1 was identified using LC/MS-MS. ( F ) Ubiquitination of MAST1 WT and K317R or/and K545R mutants upon 17-AAG treatment. ( G ) Degradation of MAST1 WT and K317R/K545R (2KR) upon 17-AAG treatment in KB-3-1 cisR and A549 cisR cells. MAST1 knockdown cells were transfected with shRNA-resistant MAST1 WT or 2KR and treated with indicated concentrations of 17-AAG for 24 hours. Data shown are representative of 2 ( A , B , and F ) and 3 ( C , D , and G ) independent biological experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: Inhibition of hsp90 induces ubiquitination of MAST1 at lysine 317/545 that leads to proteasomal degradation. ( A ) Effect of 17-AAG on MAST1 ubiquitination. 293T cells with GST-MAST1 and HA-tagged ubiquitin (Ub) were treated with 17-AAG for 4 hours and subjected to GST pull down. Anti-HA antibody was used to detect ubiquitinated MAST1. ( B and C ) Effect of 17-AAG on MAST1 proteasomal degradation. Cells were treated with or without MG-132 (10 μM) before addition of 17-AAG (1 μM) in 293T ( B ) or cisplatin-resistant cancer cells ( C ), and exogenous or endogenous MAST1 levels were detected, respectively. cRaf and AKT levels are shown for comparison. ( D ) Effect of 17-AAG on MAST1 ubiquitination and proteasomal degradation in cisplatin-resistant cancer cells. ( E ) MS spectra of ubiquitinated peptide fragments of MAST1. 293T cells with GST-MAST1 were treated with 1 μM of 17-AAG for 4 hours. Ubiquitination at K317 and K545 in MAST1 was identified using LC/MS-MS. ( F ) Ubiquitination of MAST1 WT and K317R or/and K545R mutants upon 17-AAG treatment. ( G ) Degradation of MAST1 WT and K317R/K545R (2KR) upon 17-AAG treatment in KB-3-1 cisR and A549 cisR cells. MAST1 knockdown cells were transfected with shRNA-resistant MAST1 WT or 2KR and treated with indicated concentrations of 17-AAG for 24 hours. Data shown are representative of 2 ( A , B , and F ) and 3 ( C , D , and G ) independent biological experiments.

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Inhibition, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transfection, shRNA

    CHIP degrades MAST1, which consequently sensitizes cisplatin-resistant cells to cisplatin. ( A ) Effect of CHIP overexpression and MAST1 knockdown on cisplatin sensitivity and MAST1 protein level. ( B ) Effect of MAST1 WT rescue expression on cisplatin sensitivity and MAST1 protein level in cells with CHIP overexpression and MAST1 knockdown. ( C ) Effect of CHIP and MAST1 WT or 2KR overexpression on cisplatin sensitivity and MAST1 protein level. KB-3-1 cisR and A549 cisR cells with flag-CHIP and MAST1 knockdown or WT/2KR overexpression were treated with increasing concentrations of cisplatin in the presence of 17-AAG for 48 hours. Cell viability was determined by CellTiter-Glo assay. Data are mean ± SD from 3 technical replicates and representative of 4 ( A ) and 2 ( B and C ) independent biological experiments. Statistical analysis was performed by 1-way ANOVA. *** P

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: CHIP degrades MAST1, which consequently sensitizes cisplatin-resistant cells to cisplatin. ( A ) Effect of CHIP overexpression and MAST1 knockdown on cisplatin sensitivity and MAST1 protein level. ( B ) Effect of MAST1 WT rescue expression on cisplatin sensitivity and MAST1 protein level in cells with CHIP overexpression and MAST1 knockdown. ( C ) Effect of CHIP and MAST1 WT or 2KR overexpression on cisplatin sensitivity and MAST1 protein level. KB-3-1 cisR and A549 cisR cells with flag-CHIP and MAST1 knockdown or WT/2KR overexpression were treated with increasing concentrations of cisplatin in the presence of 17-AAG for 48 hours. Cell viability was determined by CellTiter-Glo assay. Data are mean ± SD from 3 technical replicates and representative of 4 ( A ) and 2 ( B and C ) independent biological experiments. Statistical analysis was performed by 1-way ANOVA. *** P

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Chromatin Immunoprecipitation, Over Expression, Expressing, Glo Assay

    Hsp90B binds to and stabilizes MAST1 in cisplatin-resistant cancer cells. ( A ) 2D gel electrophoresis–based proteomic analysis for MAST1-interacting protein identification. 293T cells expressing GST-MAST1 or GST alone were subjected to GST pull down and eluates were separated by 2D gel electrophoresis and visualized by silver staining. Black arrow indicates hsp90B only shown in GST-MAST1 eluates. ( B ) MS spectra of hsp90B fragment identified by LC-MS/MS. ( C ) Endogenous interaction between MAST1 and hsp90B was determined by MAST1 coimmunoprecipitation in cisplatin-resistant cancer cells. ( D and E ) Interaction of hsp90 isoforms with MAST1. Interaction was determined by coimmunoprecipitation. Myc-MAST1 and flag-hsp90B or hsp90A1 were overexpressed in KB-3-1 cisR and A549 cisR cells in E . ( F ) Effect of hsp90 inhibition on MAST1 protein level. Cisplatin-resistant cancer cells were treated with increasing concentrations of 17-AAG for 24 hours. MAST1 protein levels were determined by Western blotting. Data are representative of 2 ( A and C – E ) and 3 ( F ) independent biological experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

    doi: 10.1172/JCI125963

    Figure Lengend Snippet: Hsp90B binds to and stabilizes MAST1 in cisplatin-resistant cancer cells. ( A ) 2D gel electrophoresis–based proteomic analysis for MAST1-interacting protein identification. 293T cells expressing GST-MAST1 or GST alone were subjected to GST pull down and eluates were separated by 2D gel electrophoresis and visualized by silver staining. Black arrow indicates hsp90B only shown in GST-MAST1 eluates. ( B ) MS spectra of hsp90B fragment identified by LC-MS/MS. ( C ) Endogenous interaction between MAST1 and hsp90B was determined by MAST1 coimmunoprecipitation in cisplatin-resistant cancer cells. ( D and E ) Interaction of hsp90 isoforms with MAST1. Interaction was determined by coimmunoprecipitation. Myc-MAST1 and flag-hsp90B or hsp90A1 were overexpressed in KB-3-1 cisR and A549 cisR cells in E . ( F ) Effect of hsp90 inhibition on MAST1 protein level. Cisplatin-resistant cancer cells were treated with increasing concentrations of 17-AAG for 24 hours. MAST1 protein levels were determined by Western blotting. Data are representative of 2 ( A and C – E ) and 3 ( F ) independent biological experiments.

    Article Snippet: The CHIP H260Q mutant, MAST1 variants including K317R, K545R, K317R/K545R (2KR), D497A, and CHIP or MAST1 shRNA resistant silent mutants were generated using site-directed mutagenesis kit (Agilent Technologies).

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Expressing, Silver Staining, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Inhibition, Western Blot

    Baseline plasma PGE2 is elevated in NYUHJD learning, NYUHJD progression and Pfizer cohorts. Plasma levels of PGE2 were determined using ACE EIA kit as described in Methods. The NYUHJD learning cohort (A) consists of symptomatic knee osteoarthritis (SKOA)

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    Article Title: Low-Grade Inflammation in Symptomatic Knee Osteoarthritis: Prognostic Value of Inflammatory Plasma Lipids and Peripheral Blood Leukocyte Biomarkers

    doi: 10.1002/art.39279

    Figure Lengend Snippet: Baseline plasma PGE2 is elevated in NYUHJD learning, NYUHJD progression and Pfizer cohorts. Plasma levels of PGE2 were determined using ACE EIA kit as described in Methods. The NYUHJD learning cohort (A) consists of symptomatic knee osteoarthritis (SKOA)

    Article Snippet: For the Pfizer cohort as well as subset of NYUHJD progression cohort and non-OA controls, plasma PGE2 and 15-HETE levels were measured using a two-dimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) (Agilent Technologies) interfaced to API 4000 Qtrap mass spectrometer (MDS-Sciex) operated in the multiple-reaction monitoring mode.

    Techniques: Enzyme-linked Immunosorbent Assay

    Proteins differentially expressed by HTR-8/SVneo cells cultured under serum-depleted conditions. a HTR-8/SVneo cells were grown in medium with 10 or 0 % FBS for 24 h (in triplicate). Total protein extracts (0.50 mg) were separated by 18 cm 2DE gels as previously described ( n = 3). The protein spots were analyzed via LC ESI-Q/TOF and identified using bioinformatics analysis. MW – molecular weight marker (RPN5800, GE). b Effect of serum depletion on the expression of some trophoblast proteins. Statistical analysis of 2DE protein spots identified by MS/MS in HTR-8/SVneo proteomes either with 10 % FBS or without FBS ( n = 3). Kolmogorov-Smirnov test (D value): 0.10, D > 0.642 (a, D = 1.0; b, D = 0.67)

    Journal: Cellular & Molecular Biology Letters

    Article Title: Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo

    doi: 10.1186/s11658-016-0018-9

    Figure Lengend Snippet: Proteins differentially expressed by HTR-8/SVneo cells cultured under serum-depleted conditions. a HTR-8/SVneo cells were grown in medium with 10 or 0 % FBS for 24 h (in triplicate). Total protein extracts (0.50 mg) were separated by 18 cm 2DE gels as previously described ( n = 3). The protein spots were analyzed via LC ESI-Q/TOF and identified using bioinformatics analysis. MW – molecular weight marker (RPN5800, GE). b Effect of serum depletion on the expression of some trophoblast proteins. Statistical analysis of 2DE protein spots identified by MS/MS in HTR-8/SVneo proteomes either with 10 % FBS or without FBS ( n = 3). Kolmogorov-Smirnov test (D value): 0.10, D > 0.642 (a, D = 1.0; b, D = 0.67)

    Article Snippet: LC-ESI-MS/MS analysis of tryptic peptides was performed on an Agilent Technologies Q-TOF LC-ESI-MS/MS Instrument equipped with 1200 Series liquid chromatography, chip cube ESI MS interface, and 6530 Q-TOF mass spectrometer.

    Techniques: Cell Culture, Two-Dimensional Gel Electrophoresis, Molecular Weight, Marker, Serum Depletion, Expressing, Mass Spectrometry

    Isobaric tags for relative quantification (iTRAQ) of modulated proteins in 3T3L1-PG cells. a Workflow followed for the relative quantification of modulated proteins in PG-3T3L1 cells. b Summary of the protein identification and relative quantification. c Significantly modulated biological processes after String 9.0 analysis of the modulated individual proteins. GFP green fluorescent protein, GO gene ontology, LC-MALDI-TOF liquid chromatography coupled offline to matrix-assisted laser desorption ionization–time of flight, PG progerin

    Journal: Stem Cell Research & Therapy

    Article Title: iTRAQ-based analysis of progerin expression reveals mitochondrial dysfunction, reactive oxygen species accumulation and altered proteostasis

    doi: 10.1186/s13287-015-0110-5

    Figure Lengend Snippet: Isobaric tags for relative quantification (iTRAQ) of modulated proteins in 3T3L1-PG cells. a Workflow followed for the relative quantification of modulated proteins in PG-3T3L1 cells. b Summary of the protein identification and relative quantification. c Significantly modulated biological processes after String 9.0 analysis of the modulated individual proteins. GFP green fluorescent protein, GO gene ontology, LC-MALDI-TOF liquid chromatography coupled offline to matrix-assisted laser desorption ionization–time of flight, PG progerin

    Article Snippet: iTRAQ relative quantification by two-dimensional LC-MALDI-TOF/TOF analysis In a first step, the desalted peptides (starting amount of digested peptides: 100 μg) were fractionated by basic reversed-phase extraction in a 1200 HPLC system (Agilent, Wilmington, DE, USA).

    Techniques: Liquid Chromatography