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  • 94
    Thermo Fisher phosphopeptide enrichment piercetm phosphopeptide enrichment
    Phosphopeptide Enrichment Piercetm Phosphopeptide Enrichment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore phosphopeptide enrichment kit
    Phosphopeptide Enrichment Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore phosphopeptide
    Igo1 associates and inhibits the phosphatase activity of PP2A Cdc55 in a phosphorylation dependent manner. A) Either MBP or MBP fused to Igo1, Igo-S64A and Igo1-S64D were purified from bacteria and equal amount of protein was incubated with yeast cell expressing Cdc55-TAP. The proteins bound to the beads were run on 10% SDS-PAGE and probed with anti-TAP antibody. The MBP purified proteins were visualized by coomassie staining of SDS-PAGE gels. WCE denotes whole cell extracts. B) GST-Rim15 and GST-Rim15-kd were purified from yeast cells. Equal amounts of MBP fused Igo1, Igo1-S64A and Igo1-S64D were subjected to in vitro phosphorylation using GST-Rim15 and GST-Rim15-kd respectively. MBP fused proteins were then pulled down with amylose beads and mixed with soluble protein extracts from a yeast strain expressing Cdc55-TAP. The proteins bound to the beads were analysed by western blotting using an anti-TAP antibody. Purified MBP-tagged proteins were visualized by coomassie staining of SDS-PAGE gels. Phosphorylation of Igo1 by Rim15 at S-64 was assayed using a phospho-specific antibody. WCE denotes whole cell extracts. C) TAP eluates from CDC55 -TAP and untagged strains were analysed by silver staining. D) Phosphatase activity of TAP eluates from CDC55 -TAP and untagged strains was measured using a colorimetric assay (Millipore). E) Phospho-mimetic mutant of Igo1 (Igo1S64D) inhibits the phosphatase activity of PP2A Cdc55 . Purified Cdc55 was incubated with equal amount (25 µg) of MBP-Igo1, MBP-Igo1-S64A and MBP-Igo1-S64D respectively. MBP was used as a control. The mixture was then incubated with 500 µM <t>phosphopeptide</t> (Millipore). The release of free phosphate was measured by colorimetric assay (Millipore).
    Phosphopeptide, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    PRO Scientific phosphopeptide
    Igo1 associates and inhibits the phosphatase activity of PP2A Cdc55 in a phosphorylation dependent manner. A) Either MBP or MBP fused to Igo1, Igo-S64A and Igo1-S64D were purified from bacteria and equal amount of protein was incubated with yeast cell expressing Cdc55-TAP. The proteins bound to the beads were run on 10% SDS-PAGE and probed with anti-TAP antibody. The MBP purified proteins were visualized by coomassie staining of SDS-PAGE gels. WCE denotes whole cell extracts. B) GST-Rim15 and GST-Rim15-kd were purified from yeast cells. Equal amounts of MBP fused Igo1, Igo1-S64A and Igo1-S64D were subjected to in vitro phosphorylation using GST-Rim15 and GST-Rim15-kd respectively. MBP fused proteins were then pulled down with amylose beads and mixed with soluble protein extracts from a yeast strain expressing Cdc55-TAP. The proteins bound to the beads were analysed by western blotting using an anti-TAP antibody. Purified MBP-tagged proteins were visualized by coomassie staining of SDS-PAGE gels. Phosphorylation of Igo1 by Rim15 at S-64 was assayed using a phospho-specific antibody. WCE denotes whole cell extracts. C) TAP eluates from CDC55 -TAP and untagged strains were analysed by silver staining. D) Phosphatase activity of TAP eluates from CDC55 -TAP and untagged strains was measured using a colorimetric assay (Millipore). E) Phospho-mimetic mutant of Igo1 (Igo1S64D) inhibits the phosphatase activity of PP2A Cdc55 . Purified Cdc55 was incubated with equal amount (25 µg) of MBP-Igo1, MBP-Igo1-S64A and MBP-Igo1-S64D respectively. MBP was used as a control. The mixture was then incubated with 500 µM <t>phosphopeptide</t> (Millipore). The release of free phosphate was measured by colorimetric assay (Millipore).
    Phosphopeptide, supplied by PRO Scientific, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abbiotec phosphopeptides
    AMPK inhibits YAP activity through phosphorylation of serine 94 ( a ) Phosphomimetic mutant of YAP S94 abolishes YAP activity. The indicated plasmids were co-transfected with a 5×UAS-luciferase reporter for Gal4-TEAD4 and Renilla construct into HEK293T cells. The firefly luciferase activity levels were measured and normalized to Renilla luciferase activity levels (error bars represent ± s.e.m. from n=6 biological replicates). ( b ) S94 is important for YAP activity and inhibition by AMPK. All three AMPK phosphorylation sites in YAP were mutated to alanine. The YAP plasmids were co-transfected with the 5xUAS-luciferase reporter for Gal4-TEAD4 into HEK293T cells together with the control Renilla luciferase. After 48 hr, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control (error bars represent ± s.e.m. from n=6 biological replicates). ( c ) S94 of YAP is essential for 2-DG-induced disruption of YAP-TEAD complex. HEK293A cells were transiently co-transfected with the indicated plasmids followed by treatment with 2-DG. Interaction between Flag-YAP and Myc-TEAD4 was determined by co-immunoprecipitation. ( d ) Evaluation of phospho-specific antibodies for YAP S94 phosphorylation. Phosphoantibody was prepared by immunizing rabbits with synthetic <t>phosphopeptides</t> containing phospho-YAP S94. Recombinant GST-YAP (51–270) fragment was purified from bacteria and phosphorylated by AMPK in vitro . After the reaction, 5 ng of the GST-YAP protein was detected by phospho-YAP S94 or GST antibody. The non-phosphorylatable mutant, GST-YAP-S94A was used as a negative control. ( e ) AMPK increases YAP S94 phosphorylation in transfected cells. Flag-YAP WT and YAP-S94A mutant were transfected into HEK293 cells with or without AMPK as indicated. Flag-YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. ( f ) AICAR increases YAP S94 phosphorylation in vivo . Primary hepatocytes were treated with 2 mM AICAR for 4 hr. YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. All blots shown are representatives from three independent experiments except panel e. ** P
    Phosphopeptides, supplied by Abbiotec, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Merck & Co phosphopeptide
    AMPK inhibits YAP activity through phosphorylation of serine 94 ( a ) Phosphomimetic mutant of YAP S94 abolishes YAP activity. The indicated plasmids were co-transfected with a 5×UAS-luciferase reporter for Gal4-TEAD4 and Renilla construct into HEK293T cells. The firefly luciferase activity levels were measured and normalized to Renilla luciferase activity levels (error bars represent ± s.e.m. from n=6 biological replicates). ( b ) S94 is important for YAP activity and inhibition by AMPK. All three AMPK phosphorylation sites in YAP were mutated to alanine. The YAP plasmids were co-transfected with the 5xUAS-luciferase reporter for Gal4-TEAD4 into HEK293T cells together with the control Renilla luciferase. After 48 hr, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control (error bars represent ± s.e.m. from n=6 biological replicates). ( c ) S94 of YAP is essential for 2-DG-induced disruption of YAP-TEAD complex. HEK293A cells were transiently co-transfected with the indicated plasmids followed by treatment with 2-DG. Interaction between Flag-YAP and Myc-TEAD4 was determined by co-immunoprecipitation. ( d ) Evaluation of phospho-specific antibodies for YAP S94 phosphorylation. Phosphoantibody was prepared by immunizing rabbits with synthetic <t>phosphopeptides</t> containing phospho-YAP S94. Recombinant GST-YAP (51–270) fragment was purified from bacteria and phosphorylated by AMPK in vitro . After the reaction, 5 ng of the GST-YAP protein was detected by phospho-YAP S94 or GST antibody. The non-phosphorylatable mutant, GST-YAP-S94A was used as a negative control. ( e ) AMPK increases YAP S94 phosphorylation in transfected cells. Flag-YAP WT and YAP-S94A mutant were transfected into HEK293 cells with or without AMPK as indicated. Flag-YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. ( f ) AICAR increases YAP S94 phosphorylation in vivo . Primary hepatocytes were treated with 2 mM AICAR for 4 hr. YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. All blots shown are representatives from three independent experiments except panel e. ** P
    Phosphopeptide, supplied by Merck & Co, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript phosphopeptides
    AMPK inhibits YAP activity through phosphorylation of serine 94 ( a ) Phosphomimetic mutant of YAP S94 abolishes YAP activity. The indicated plasmids were co-transfected with a 5×UAS-luciferase reporter for Gal4-TEAD4 and Renilla construct into HEK293T cells. The firefly luciferase activity levels were measured and normalized to Renilla luciferase activity levels (error bars represent ± s.e.m. from n=6 biological replicates). ( b ) S94 is important for YAP activity and inhibition by AMPK. All three AMPK phosphorylation sites in YAP were mutated to alanine. The YAP plasmids were co-transfected with the 5xUAS-luciferase reporter for Gal4-TEAD4 into HEK293T cells together with the control Renilla luciferase. After 48 hr, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control (error bars represent ± s.e.m. from n=6 biological replicates). ( c ) S94 of YAP is essential for 2-DG-induced disruption of YAP-TEAD complex. HEK293A cells were transiently co-transfected with the indicated plasmids followed by treatment with 2-DG. Interaction between Flag-YAP and Myc-TEAD4 was determined by co-immunoprecipitation. ( d ) Evaluation of phospho-specific antibodies for YAP S94 phosphorylation. Phosphoantibody was prepared by immunizing rabbits with synthetic <t>phosphopeptides</t> containing phospho-YAP S94. Recombinant GST-YAP (51–270) fragment was purified from bacteria and phosphorylated by AMPK in vitro . After the reaction, 5 ng of the GST-YAP protein was detected by phospho-YAP S94 or GST antibody. The non-phosphorylatable mutant, GST-YAP-S94A was used as a negative control. ( e ) AMPK increases YAP S94 phosphorylation in transfected cells. Flag-YAP WT and YAP-S94A mutant were transfected into HEK293 cells with or without AMPK as indicated. Flag-YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. ( f ) AICAR increases YAP S94 phosphorylation in vivo . Primary hepatocytes were treated with 2 mM AICAR for 4 hr. YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. All blots shown are representatives from three independent experiments except panel e. ** P
    Phosphopeptides, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AnaSpec phosphopeptides
    Detection of MS-identified GRIP1 phosphorylation sites using phosphospecific antibodies. (A) U2OS-rGR cells were untreated or treated with dex for 2 h, and the extracts were subjected to immunoblotting using 1:3,000 dilutions of the indicated antisera in the absence or presence the relevant blocking <t>phosphopeptides.</t> pep, peptide; α, anti. (B to D) U2OS-rGR (B), A549 (C), or primary mouse bone marrow-derived macrophages (D) were treated with dex for indicated periods of time, and whole-cell extracts were prepared. Immunoblotting was performed with antibodies to total GRIP1, antisera to indicated phosphosites, and STAT3 to confirm equal loading.
    Phosphopeptides, supplied by AnaSpec, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GL Biochem phosphopeptides
    SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target. SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted <t>phosphopeptides</t> (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the K D values for the respective phosphopeptides. Binding analysis of ( A ) JAK, Shc-1, or wild-type and ( B ) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective K D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. ( C ) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. ( D ) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).
    Phosphopeptides, supplied by GL Biochem, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Protea Bio phosphopeptide standard
    MS 3 trend plots for two phosphoserine modified peptides based on treatment group. <t>Phosphopeptide</t> pS 991 (MHLP pS PTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNF pY R ) shows similar trends. These data are similar to trends observed for pY peptide MHLPSPTDSNF pY R. Phosphopeptide pS 1166 (GSHQI pS LDNPDYQQDFFPK) normalized to an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPD pY QQDFFP K ) show similar trends. These data show that MS 3 measurements can be used for quantification of protein modifications. These data represent three technical injects of biological replicate 2. The underlined amino acid indicates which amino acid was stable-isotope labeled.
    Phosphopeptide Standard, supplied by Protea Bio, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Peptron Inc phosphopeptide column
    Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of <t>GSK3β-non-phosphopeptide</t> ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.
    Phosphopeptide Column, supplied by Peptron Inc, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH rii phosphopeptide
    Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of <t>GSK3β-non-phosphopeptide</t> ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.
    Rii Phosphopeptide, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 88/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GenScript phosphopeptide dtaslsttpsespr
    Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of <t>GSK3β-non-phosphopeptide</t> ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.
    Phosphopeptide Dtaslsttpsespr, supplied by GenScript, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GL Sciences phosphopeptide enrichment
    Optimization of TiO 2 <t>phosphopeptide</t> enrichment using 0.5 mg of rat brain lysate and various additives to reduce the binding of non-phosphopeptides. Peptide-to-bead ratio is 1:4 (w/w). (A) The number and percentage of identified phosphopeptides without
    Phosphopeptide Enrichment, supplied by GL Sciences, used in various techniques. Bioz Stars score: 92/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioGenes GmbH phosphopeptide ks
    Optimization of TiO 2 <t>phosphopeptide</t> enrichment using 0.5 mg of rat brain lysate and various additives to reduce the binding of non-phosphopeptides. Peptide-to-bead ratio is 1:4 (w/w). (A) The number and percentage of identified phosphopeptides without
    Phosphopeptide Ks, supplied by BioGenes GmbH, 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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    Promega phosphopeptide substrate
    AKAP79 and cyclosporin bind to a common surface on PP2B. ( A ) GST-pulldown experiments using N- and C- fragments of AKAP79 and testing for competition using cyclosporin/cyclophilin complexes, n = 3. ( B ) Quantification of PP2B signals in GST-pulldowns, normalized to lane 2, n = 3. Data are represented as mean ± SEM. ( C ) Phosphatase activity assay on samples from lanes 1–4 using a <t>phosphopeptide</t> substrate, n = 4. Data are represented as mean ± SEM.
    Phosphopeptide Substrate, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals small phosphopeptides
    AKAP79 and cyclosporin bind to a common surface on PP2B. ( A ) GST-pulldown experiments using N- and C- fragments of AKAP79 and testing for competition using cyclosporin/cyclophilin complexes, n = 3. ( B ) Quantification of PP2B signals in GST-pulldowns, normalized to lane 2, n = 3. Data are represented as mean ± SEM. ( C ) Phosphatase activity assay on samples from lanes 1–4 using a <t>phosphopeptide</t> substrate, n = 4. Data are represented as mean ± SEM.
    Small Phosphopeptides, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PhosphoSolutions phosphopeptide columns
    Detection of S129 Phosphorylation In Vivo by <t>Phosphopeptide</t> Mapping and S129-Specific Phosphoantibodies
    Phosphopeptide Columns, supplied by PhosphoSolutions, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phosphopeptide immunogen
    Detection of S129 Phosphorylation In Vivo by <t>Phosphopeptide</t> Mapping and S129-Specific Phosphoantibodies
    Phosphopeptide Immunogen, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphopeptide levels
    Detection of S129 Phosphorylation In Vivo by <t>Phosphopeptide</t> Mapping and S129-Specific Phosphoantibodies
    Phosphopeptide Levels, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk3β phosphopeptide sequences
    Detection of S129 Phosphorylation In Vivo by <t>Phosphopeptide</t> Mapping and S129-Specific Phosphoantibodies
    Gsk3β Phosphopeptide Sequences, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa phosphopeptide enrichment spin column
    Detection of S129 Phosphorylation In Vivo by <t>Phosphopeptide</t> Mapping and S129-Specific Phosphoantibodies
    Phosphopeptide Enrichment Spin Column, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Igo1 associates and inhibits the phosphatase activity of PP2A Cdc55 in a phosphorylation dependent manner. A) Either MBP or MBP fused to Igo1, Igo-S64A and Igo1-S64D were purified from bacteria and equal amount of protein was incubated with yeast cell expressing Cdc55-TAP. The proteins bound to the beads were run on 10% SDS-PAGE and probed with anti-TAP antibody. The MBP purified proteins were visualized by coomassie staining of SDS-PAGE gels. WCE denotes whole cell extracts. B) GST-Rim15 and GST-Rim15-kd were purified from yeast cells. Equal amounts of MBP fused Igo1, Igo1-S64A and Igo1-S64D were subjected to in vitro phosphorylation using GST-Rim15 and GST-Rim15-kd respectively. MBP fused proteins were then pulled down with amylose beads and mixed with soluble protein extracts from a yeast strain expressing Cdc55-TAP. The proteins bound to the beads were analysed by western blotting using an anti-TAP antibody. Purified MBP-tagged proteins were visualized by coomassie staining of SDS-PAGE gels. Phosphorylation of Igo1 by Rim15 at S-64 was assayed using a phospho-specific antibody. WCE denotes whole cell extracts. C) TAP eluates from CDC55 -TAP and untagged strains were analysed by silver staining. D) Phosphatase activity of TAP eluates from CDC55 -TAP and untagged strains was measured using a colorimetric assay (Millipore). E) Phospho-mimetic mutant of Igo1 (Igo1S64D) inhibits the phosphatase activity of PP2A Cdc55 . Purified Cdc55 was incubated with equal amount (25 µg) of MBP-Igo1, MBP-Igo1-S64A and MBP-Igo1-S64D respectively. MBP was used as a control. The mixture was then incubated with 500 µM phosphopeptide (Millipore). The release of free phosphate was measured by colorimetric assay (Millipore).

    Journal: PLoS Genetics

    Article Title: The Rim15-Endosulfine-PP2ACdc55 Signalling Module Regulates Entry into Gametogenesis and Quiescence via Distinct Mechanisms in Budding Yeast

    doi: 10.1371/journal.pgen.1004456

    Figure Lengend Snippet: Igo1 associates and inhibits the phosphatase activity of PP2A Cdc55 in a phosphorylation dependent manner. A) Either MBP or MBP fused to Igo1, Igo-S64A and Igo1-S64D were purified from bacteria and equal amount of protein was incubated with yeast cell expressing Cdc55-TAP. The proteins bound to the beads were run on 10% SDS-PAGE and probed with anti-TAP antibody. The MBP purified proteins were visualized by coomassie staining of SDS-PAGE gels. WCE denotes whole cell extracts. B) GST-Rim15 and GST-Rim15-kd were purified from yeast cells. Equal amounts of MBP fused Igo1, Igo1-S64A and Igo1-S64D were subjected to in vitro phosphorylation using GST-Rim15 and GST-Rim15-kd respectively. MBP fused proteins were then pulled down with amylose beads and mixed with soluble protein extracts from a yeast strain expressing Cdc55-TAP. The proteins bound to the beads were analysed by western blotting using an anti-TAP antibody. Purified MBP-tagged proteins were visualized by coomassie staining of SDS-PAGE gels. Phosphorylation of Igo1 by Rim15 at S-64 was assayed using a phospho-specific antibody. WCE denotes whole cell extracts. C) TAP eluates from CDC55 -TAP and untagged strains were analysed by silver staining. D) Phosphatase activity of TAP eluates from CDC55 -TAP and untagged strains was measured using a colorimetric assay (Millipore). E) Phospho-mimetic mutant of Igo1 (Igo1S64D) inhibits the phosphatase activity of PP2A Cdc55 . Purified Cdc55 was incubated with equal amount (25 µg) of MBP-Igo1, MBP-Igo1-S64A and MBP-Igo1-S64D respectively. MBP was used as a control. The mixture was then incubated with 500 µM phosphopeptide (Millipore). The release of free phosphate was measured by colorimetric assay (Millipore).

    Article Snippet: 25 µg of purified MBP, MBP-Igo1, MBP-Igo1S64A and MBP-Igo1S64D was incubated with 500 µM phosphopeptide (Millipore).

    Techniques: Activity Assay, Purification, Incubation, Expressing, SDS Page, Staining, In Vitro, Western Blot, Silver Staining, Colorimetric Assay, Mutagenesis

    AMPK inhibits YAP activity through phosphorylation of serine 94 ( a ) Phosphomimetic mutant of YAP S94 abolishes YAP activity. The indicated plasmids were co-transfected with a 5×UAS-luciferase reporter for Gal4-TEAD4 and Renilla construct into HEK293T cells. The firefly luciferase activity levels were measured and normalized to Renilla luciferase activity levels (error bars represent ± s.e.m. from n=6 biological replicates). ( b ) S94 is important for YAP activity and inhibition by AMPK. All three AMPK phosphorylation sites in YAP were mutated to alanine. The YAP plasmids were co-transfected with the 5xUAS-luciferase reporter for Gal4-TEAD4 into HEK293T cells together with the control Renilla luciferase. After 48 hr, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control (error bars represent ± s.e.m. from n=6 biological replicates). ( c ) S94 of YAP is essential for 2-DG-induced disruption of YAP-TEAD complex. HEK293A cells were transiently co-transfected with the indicated plasmids followed by treatment with 2-DG. Interaction between Flag-YAP and Myc-TEAD4 was determined by co-immunoprecipitation. ( d ) Evaluation of phospho-specific antibodies for YAP S94 phosphorylation. Phosphoantibody was prepared by immunizing rabbits with synthetic phosphopeptides containing phospho-YAP S94. Recombinant GST-YAP (51–270) fragment was purified from bacteria and phosphorylated by AMPK in vitro . After the reaction, 5 ng of the GST-YAP protein was detected by phospho-YAP S94 or GST antibody. The non-phosphorylatable mutant, GST-YAP-S94A was used as a negative control. ( e ) AMPK increases YAP S94 phosphorylation in transfected cells. Flag-YAP WT and YAP-S94A mutant were transfected into HEK293 cells with or without AMPK as indicated. Flag-YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. ( f ) AICAR increases YAP S94 phosphorylation in vivo . Primary hepatocytes were treated with 2 mM AICAR for 4 hr. YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. All blots shown are representatives from three independent experiments except panel e. ** P

    Journal: Nature cell biology

    Article Title: Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway

    doi: 10.1038/ncb3111

    Figure Lengend Snippet: AMPK inhibits YAP activity through phosphorylation of serine 94 ( a ) Phosphomimetic mutant of YAP S94 abolishes YAP activity. The indicated plasmids were co-transfected with a 5×UAS-luciferase reporter for Gal4-TEAD4 and Renilla construct into HEK293T cells. The firefly luciferase activity levels were measured and normalized to Renilla luciferase activity levels (error bars represent ± s.e.m. from n=6 biological replicates). ( b ) S94 is important for YAP activity and inhibition by AMPK. All three AMPK phosphorylation sites in YAP were mutated to alanine. The YAP plasmids were co-transfected with the 5xUAS-luciferase reporter for Gal4-TEAD4 into HEK293T cells together with the control Renilla luciferase. After 48 hr, the firefly luciferase activity was measured and normalized to the co-transfected Renilla luciferase internal control (error bars represent ± s.e.m. from n=6 biological replicates). ( c ) S94 of YAP is essential for 2-DG-induced disruption of YAP-TEAD complex. HEK293A cells were transiently co-transfected with the indicated plasmids followed by treatment with 2-DG. Interaction between Flag-YAP and Myc-TEAD4 was determined by co-immunoprecipitation. ( d ) Evaluation of phospho-specific antibodies for YAP S94 phosphorylation. Phosphoantibody was prepared by immunizing rabbits with synthetic phosphopeptides containing phospho-YAP S94. Recombinant GST-YAP (51–270) fragment was purified from bacteria and phosphorylated by AMPK in vitro . After the reaction, 5 ng of the GST-YAP protein was detected by phospho-YAP S94 or GST antibody. The non-phosphorylatable mutant, GST-YAP-S94A was used as a negative control. ( e ) AMPK increases YAP S94 phosphorylation in transfected cells. Flag-YAP WT and YAP-S94A mutant were transfected into HEK293 cells with or without AMPK as indicated. Flag-YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. ( f ) AICAR increases YAP S94 phosphorylation in vivo . Primary hepatocytes were treated with 2 mM AICAR for 4 hr. YAP was immunoprecipitated and phosphorylation of S94 was detected by the pYAP(S94) phosphoantibody. All blots shown are representatives from three independent experiments except panel e. ** P

    Article Snippet: Anti-phosphorylated S94 antibody was generated by immunizing rabbits with phosphopeptides (Abbiotec and Gentex, 1:500).

    Techniques: Activity Assay, Mutagenesis, Transfection, Luciferase, Construct, Inhibition, Immunoprecipitation, Recombinant, Purification, In Vitro, Negative Control, In Vivo

    Detection of MS-identified GRIP1 phosphorylation sites using phosphospecific antibodies. (A) U2OS-rGR cells were untreated or treated with dex for 2 h, and the extracts were subjected to immunoblotting using 1:3,000 dilutions of the indicated antisera in the absence or presence the relevant blocking phosphopeptides. pep, peptide; α, anti. (B to D) U2OS-rGR (B), A549 (C), or primary mouse bone marrow-derived macrophages (D) were treated with dex for indicated periods of time, and whole-cell extracts were prepared. Immunoblotting was performed with antibodies to total GRIP1, antisera to indicated phosphosites, and STAT3 to confirm equal loading.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Dependent Phosphorylation of the Transcriptional Coregulator GRIP1

    doi: 10.1128/MCB.06473-11

    Figure Lengend Snippet: Detection of MS-identified GRIP1 phosphorylation sites using phosphospecific antibodies. (A) U2OS-rGR cells were untreated or treated with dex for 2 h, and the extracts were subjected to immunoblotting using 1:3,000 dilutions of the indicated antisera in the absence or presence the relevant blocking phosphopeptides. pep, peptide; α, anti. (B to D) U2OS-rGR (B), A549 (C), or primary mouse bone marrow-derived macrophages (D) were treated with dex for indicated periods of time, and whole-cell extracts were prepared. Immunoblotting was performed with antibodies to total GRIP1, antisera to indicated phosphosites, and STAT3 to confirm equal loading.

    Article Snippet: The phosphopeptides C+GSNYALKMN(pS469)PSQSSP-NH2, C+NPGQPTSML(pS487)PRHR-NH2, PRHRM(pS493)PGVAGSPRI+C-NH2, and PGVAG(pS499)PRIPPSQFS+C-NH2 were synthesized and purified by high-pressure liquid chromatography (HPLC) by Anaspec.

    Techniques: Mass Spectrometry, Blocking Assay, Derivative Assay

    SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target. SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the K D values for the respective phosphopeptides. Binding analysis of ( A ) JAK, Shc-1, or wild-type and ( B ) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective K D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. ( C ) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. ( D ) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

    doi: 10.1371/journal.pone.0070536

    Figure Lengend Snippet: SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target. SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the K D values for the respective phosphopeptides. Binding analysis of ( A ) JAK, Shc-1, or wild-type and ( B ) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective K D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. ( C ) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. ( D ) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).

    Article Snippet: All phosphopeptides were purchased from GL Biochem, China.

    Techniques: Binding Assay, SPR Assay, Recombinant, Concentration Assay, Transfection, Western Blot

    MS 3 trend plots for two phosphoserine modified peptides based on treatment group. Phosphopeptide pS 991 (MHLP pS PTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNF pY R ) shows similar trends. These data are similar to trends observed for pY peptide MHLPSPTDSNF pY R. Phosphopeptide pS 1166 (GSHQI pS LDNPDYQQDFFPK) normalized to an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPD pY QQDFFP K ) show similar trends. These data show that MS 3 measurements can be used for quantification of protein modifications. These data represent three technical injects of biological replicate 2. The underlined amino acid indicates which amino acid was stable-isotope labeled.

    Journal: Journal of Proteome Research

    Article Title: Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides

    doi: 10.1021/pr201240a

    Figure Lengend Snippet: MS 3 trend plots for two phosphoserine modified peptides based on treatment group. Phosphopeptide pS 991 (MHLP pS PTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNF pY R ) shows similar trends. These data are similar to trends observed for pY peptide MHLPSPTDSNF pY R. Phosphopeptide pS 1166 (GSHQI pS LDNPDYQQDFFPK) normalized to an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPD pY QQDFFP K ) show similar trends. These data show that MS 3 measurements can be used for quantification of protein modifications. These data represent three technical injects of biological replicate 2. The underlined amino acid indicates which amino acid was stable-isotope labeled.

    Article Snippet: Synthetic phosphorylated peptides, DRVpY IHPF and IKNLQpS LDPSH, were purchased as part of the Phosphopeptide Standard I from Protea Biosciences (Morgantown, WV).

    Techniques: Mass Spectrometry, Modification, Labeling

    Median CV across technical replicates plotted against amount of spiked in phosphorylated peptide standards. Data point colors correspond to reference peptide used for normalization. The median CVs decrease as the amount of phosphopeptide spiked in background (BSA digest) increases.

    Journal: Journal of Proteome Research

    Article Title: Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides

    doi: 10.1021/pr201240a

    Figure Lengend Snippet: Median CV across technical replicates plotted against amount of spiked in phosphorylated peptide standards. Data point colors correspond to reference peptide used for normalization. The median CVs decrease as the amount of phosphopeptide spiked in background (BSA digest) increases.

    Article Snippet: Synthetic phosphorylated peptides, DRVpY IHPF and IKNLQpS LDPSH, were purchased as part of the Phosphopeptide Standard I from Protea Biosciences (Morgantown, WV).

    Techniques:

    Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of GSK3β-non-phosphopeptide ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) *

    doi: 10.1074/jbc.M114.594952

    Figure Lengend Snippet: Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of GSK3β-non-phosphopeptide ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.

    Article Snippet: A phosphospecific GSK3β (Thr(P)356 -GSK3β) antibody to a synthetic phosphopeptide (352 NGRDpTPALFN361 ) was generated and affinity-purified first with a cognate non-phosphopeptide (NGRDTPALFN) affinity column and then with a phosphopeptide column (Peptron, Korea).

    Techniques: Expressing, Purification, SDS Page, Transfection, Western Blot, Competitive Binding Assay

    Optimization of TiO 2 phosphopeptide enrichment using 0.5 mg of rat brain lysate and various additives to reduce the binding of non-phosphopeptides. Peptide-to-bead ratio is 1:4 (w/w). (A) The number and percentage of identified phosphopeptides without

    Journal: Proteomics

    Article Title: Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome

    doi: 10.1002/pmic.201400171

    Figure Lengend Snippet: Optimization of TiO 2 phosphopeptide enrichment using 0.5 mg of rat brain lysate and various additives to reduce the binding of non-phosphopeptides. Peptide-to-bead ratio is 1:4 (w/w). (A) The number and percentage of identified phosphopeptides without

    Article Snippet: Phosphopeptide enrichment was carried out by TiO2 beads (GL sciences).

    Techniques: Binding Assay

    Effect of peptide-to-bead ratio on the selectivity of phosphopeptide enrichment. The LCMS/MS analysis was repeated with the average of relative standard deviation

    Journal: Proteomics

    Article Title: Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome

    doi: 10.1002/pmic.201400171

    Figure Lengend Snippet: Effect of peptide-to-bead ratio on the selectivity of phosphopeptide enrichment. The LCMS/MS analysis was repeated with the average of relative standard deviation

    Article Snippet: Phosphopeptide enrichment was carried out by TiO2 beads (GL sciences).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Standard Deviation

    AKAP79 and cyclosporin bind to a common surface on PP2B. ( A ) GST-pulldown experiments using N- and C- fragments of AKAP79 and testing for competition using cyclosporin/cyclophilin complexes, n = 3. ( B ) Quantification of PP2B signals in GST-pulldowns, normalized to lane 2, n = 3. Data are represented as mean ± SEM. ( C ) Phosphatase activity assay on samples from lanes 1–4 using a phosphopeptide substrate, n = 4. Data are represented as mean ± SEM.

    Journal: eLife

    Article Title: Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity

    doi: 10.7554/eLife.30872

    Figure Lengend Snippet: AKAP79 and cyclosporin bind to a common surface on PP2B. ( A ) GST-pulldown experiments using N- and C- fragments of AKAP79 and testing for competition using cyclosporin/cyclophilin complexes, n = 3. ( B ) Quantification of PP2B signals in GST-pulldowns, normalized to lane 2, n = 3. Data are represented as mean ± SEM. ( C ) Phosphatase activity assay on samples from lanes 1–4 using a phosphopeptide substrate, n = 4. Data are represented as mean ± SEM.

    Article Snippet: After the final wash, PP2B activity buffer (Promega) was added and a phosphopeptide substrate (Promega) was included to measure phosphatase activity in these samples.

    Techniques: Phosphatase Assay

    Detection of S129 Phosphorylation In Vivo by Phosphopeptide Mapping and S129-Specific Phosphoantibodies

    Journal:

    Article Title: Receptor for RACK1 Mediates Activation of JNK by Protein Kinase C

    doi: 10.1016/j.molcel.2005.06.025

    Figure Lengend Snippet: Detection of S129 Phosphorylation In Vivo by Phosphopeptide Mapping and S129-Specific Phosphoantibodies

    Article Snippet: Phosphoantibodies to S129 of JNK (p-JNKS129 ) were generated by immunizing rabbits with the peptide DANLCQVIHMELD HERMSP YLLYQMLCGIKHLHSAG followed by selective purification on phosphopeptide columns (PhosphoSolutions, Aurora, CO).

    Techniques: In Vivo