Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ C fragment are important for protecting cell from TNF-induced necroptosis via interaction with RIP1. a Immunoprecipitation of HT-29 cells treated with TSZ for the indicated times. Cell lysates were immunoprecipitated with anti-RARγ antibody and analyzed by immunoblotting with the indicated antibodies. b HEK293T cells were co-transfected with wild-type V5-RARγ (WT) or the V5-RARγ-NLSmut (NLS) and with or without FLAG-RIP1 plasmids. Cell lysates were immunoprecipitated using anti-FLAG (RIP1) antibody and analyzed with the indicated antibodies. c Scheme of RARγ and RIP1 genes ( upper panel ). HEK293T cells were co-transfected with different fragment of RARγ-V5 (F: full; N:1–201aa; C: 188–454aa) and with or without FLAG-RIP1 plasmids; or with different fragment of His-Xpress-RIP1 (F: full; N: 1–324aa; C: 324–671aa; DD: 560–669aa) and with or without RARγ-V5. Cell lysates were immunoprecipitated using anti-FLAG (RIP1) or anti-V5 (RARγ) antibody and analyzed with the indicated antibodies. d HT-29 RARγ-shRNA-A cells infected with rRARγ-C. Cell death analysis of HT-29 cont-shRNA, RARγ-shRNA-A, and RARγ-shRNA-A + rRARγ-C when treated with TSZ for 24 h was determined by PI staining using flow cytometry ( upper left panel ). (* P < 0.05 versus cont-shRNA; # P < 0.05 versus RARγ-shRNA-A; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel ( upper right panel ). Confocal microscopy of HT-29 cells infected with GFP-RARγ-C plasmid ( lower left panel ) ( blue : DAPI; green : RARγ). Western blot analysis of HEK293T cells were transfected with p-EGFP, p-EGFP-RARγ and p-EGFP-RARγ-C by using anti-GFP and anti-Actin antibodies ( lower right panel ). All blots and images above are representative of one of three experiments

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Immunoprecipitation, Western Blot, Transfection, shRNA, Infection, Staining, Flow Cytometry, Confocal Microscopy, Plasmid Preparation

Journal: Nature Communications

Article Title: The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited

doi: 10.1038/s41467-017-00496-6

Figure Lengend Snippet: RARγ initiates the formation of death complexes by dissociating RIP1 from TNFR1. a Immunoprecipitation of HT-29 cont-shRNA, TRADD-shRNA or RIP1-shRNA treated with TSZ for the indicated times. Cell lysates were immunoprecipitated using anti-TNFR1 antibody and immunoblotted with the indicated antibodies. b Sequential immunoprecipitation of HEK293T cells co-transfected with FLAG-TNFR1, DsRed-RIP1 and increasing amounts of V5-RARγ-NLSmut plasmids as indicated. First IP : cell lysates were immunoprecipitated with anti-FLAG (TNFR1) antibody. Second IP : the remaining lysates were then immunoprecipitated with anti-V5 (RARγ) antibody. The immunoprecipitated complexes were analyzed by with the indicated antibodies. c Sequential immunoprecipitation of HEK293T cells co-transfected with V5-RARγ-NLSmut, RIP1-Myc, and increasing amounts of DsRed-RIP3 plasmids as indicated. First IP : cell lysates were immunoprecipitated with anti-V5 (RARγ) antibody. Second IP : the remaining lysates were then immunoprecipitated with anti-DsRed (RIP3) antibody. The immunoprecipitated complexes were analyzed with the indicated antibodies. d WT and RIP3−/− MEFs treated with TSZ for the indicated times. Cell lysates were immunoprecipitated using anti-Caspase-8 antibody and immunoblotted with the indicated antibodies. All blots above are representative of one of three experiments

Article Snippet: Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

Techniques: Immunoprecipitation, shRNA, Transfection

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B physically interacts with GTP-bound mutant KRAS and SOS. (a) Mass spectrometry analysis of eIF2B and KRAS interactions. (Left panel) eIF2B subunits specifically interact with the G12V mutant of FLAG-KRAS 4B, but not with the G12V mutant of FLAG-KRAS 4A. (Right panel) Mutations in the C-terminus hypervariable region of FLAG-KRAS G12V impair its interaction with the eIF2B subunits. V12, G12V mutation of KRAS 4B; CS, C→S mutation in the C-terminal CAAX motif of KRAS G12V; KQ, K→Q mutations in the poly-lysine stretch of KRAS G12V; Ctrl, mass spectrometry analysis of proteins bound to a non-target antibody. ( b , c ) HEK293T cells were co-transfected with MYC-tagged constructs for eIF2Bε (panel b) or all eIF2B subunits (panel c), together with KRAS G12V variants containing C→S and polyK→polyQ mutations within the HVR (panel b) as well as various KRAS mutants harboring substitutions at G12, G13, or Q61 (panel c). Cell lysates were subjected to IP with an anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-MYC antibodies to detect KRAS and eIF2B, respectively. Lysates from HEK293T cells transfected with insert less vector DNA served as negative controls. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE) ( d ) Interaction between endogenous eIF2B, SOS and KRAS in H358 and H1703 cells in co-IP assay with antibodies against the eIF2Bε subunit. Proteins were analyzed by immunoblotting to detect endogenous KRAS, SOS and eIF2Bε. Rabbit IgG was used as a negative control. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls. ( e , f ) Cell lysates from H358 and H1703 cells were subjected to pull-down assays with GST-RBD of RAF. In panel (e), lysates from SOS1/2-proficient and SOS1/2 KD H358 cells were analyzed for bound KRAS, eIF2Bε, and SOS1 by immunoblotting. In panel (f), lysates from eIF2Bε-proficient and eIF2Bε-KD H358 (KRAS G12C) and H1703 (wild-type KRAS) cells were processed similarly using GST-RBD to detect KRAS, eIF2Bε, and SOS1. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls.

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: Mutagenesis, Mass Spectrometry, Transfection, Construct, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Negative Control

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: In silico assembly and biochemical mapping of the eIF2B:SOS:KRAS complex. (a) Putative model of the eIF2B:SOS:KRAS G12V structural assembly. eIF2B is predicted to associate either to the allosteric binding site of SOS or to GTP-bound RAS (dimer or oligomer) via its eIF2Bε subunit. In the close-up views of the predicted eIF2Bε interaction sites, the location of the mutated residues utilized in this study are highlighted with spheres on their Cα-atoms (eIF2Bε: blue spheres; SOS: orange spheres). For further information on the model see Suppl Figure 3. (b) The eIF2Bε subunit mediates the interaction of eIF2B with SOS and mutant KRAS. HEK293T cells were co-transfected with MYC-tagged constructs for each eIF2B subunit separately, HA-SOS1 and FLAG-KRAS G12V. Cell lysates were subjected to IP with an anti-MYC antibody, followed by immunoblotting with anti-FLAG, anti-HA, and anti-MYC antibodies to detect KRAS, SOS1, and eIF2B subunits, respectively. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE). (c) SOS residues 566–1046 (SOS CAT ) interacts with eIF2Bε and mutant KRAS, but to a lesser extent wild type (WT) KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, T7-tagged SOS CAT , and either FLAG-tagged KRAS G12V or WT KRAS. Cell lysates were co-IPed using anti-MYC or anti-FLAG antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies. (d) Mutations in the allosteric RAS-binding site of SOS CAT impair the interaction with eIF2Bε and mutant KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, FLAG-tagged KRAS G12V and T7-tagged SOS CAT either wild type or containing the W729E or L687E/R688A mutations. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies to detect the respective proteins. (e) The catalytic GEF activity of eIF2Bε is essential for its interaction with SOS and mutant KRAS. HEK293T cells were transfected with HA-SOS1, FLAG-KRAS G12V and MYC-eIF2Bε either wild-type or carrying the hyperactive D154A mutation, the catalytically inactive N263K mutation, or the QVA→ISP mutation in the C-terminus. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. (f) Mutations in eIF2Bε impair its interaction with SOS and mutant KRAS. HEK293T cells were transfected with FLAG-tagged KRAS G12V, HA-tagged SOS1, and MYC-tagged eIF2Bε, either wild-type or containing the K103E, K141E, or K103E/K141E mutations. Cell lysates were subjected to co-IP with anti-FLAG or anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. ( b - f ) Data represent one of three reproducible experiments.

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: In Silico, Binding Assay, Mutagenesis, Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2Bε co-localizes with SOS and mutant KRAS at the PM. (a) IF analysis of MYC-eIF2Bε (red, 568 nm), HA-SOS1 (blue) and FLAG-KRAS (green), either WT (left) or G12V (right), co-expressed in HEK293T cells. MYC-tagged eIF2Bε was expressed in HEK293T cells either as wild-type or in mutant forms, including the hyperactive D154A mutant, the catalytically inactive N263K mutant, or the QVA→ISP mutant that disrupts interaction with SOS. Protein localization was assessed by confocal microscopy. The white signal in the merged images indicates the co-localization of the three proteins at the periphery of the cell. (b) IF analysis of cells described in panel (a) assessing PM localization of FLAG-tagged KRAS (green) following co-staining with Cholera Toxin subunit B (CT-B, far-red, 647 nm), a PM marker. Mander’s colocalization coefficients, analyzed with JACoP (ImageJ), were used to quantify the extent of FLAG-KRAS colocalization with CT-B at the PM. Scale bar: 5 μm. The yellow signal in the images indicates the co-localization of FLAG-KRAS and CT-B at the PM. (c) H358 cells (KRAS G12C) and H1703 cells (wild-type KRAS) expressing either scrambled shRNA or eIF2Bε shRNA were fractionated into nuclear (N), cytosolic (C), organelle (O), and plasma membrane (PM) fractions. Each fraction was subjected to immunoblotting to detect SOS1, eIF2Bε, and KRAS. Fractionation quality was verified using specific markers: EGFR for the PM, THOC1 for the nucleus, and α-TUBULIN for the cytosol.

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: Mutagenesis, Confocal Microscopy, IF-cells, Staining, Marker, Expressing, shRNA, Clinical Proteomics, Membrane, Western Blot, Fractionation

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports mutant KRAS PM localization and nanoclustering via the GSL pathway. (a) eIF2B specifically promotes mutant KRAS localization at PM. Representative confocal images of T47D cells expressing either GFP-KRAS G12V or GFP-HRAS G12V, treated with either scrambled shRNA or eIF2Bε shRNA. Cells were stained with CellMask to label the PM. Co-localization of GFP-KRAS with CellMask was quantified using Manders’ coefficient and is presented as mean ± SEM (n = 3). Scale bar: 10 μm (b) eIF2B controls the localization and spatial organization of mutant KRAS at the PM. PM sheets were isolated from H1703 cells stably expressing GFP-KRAS G12C and transfected with either scrambled or eIF2Bε siRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized by EM. Representative EM images are shown. Quantification of gold particles is presented as mean number ± SEM (n = 32). Spatial distribution was analyzed, and L max values, indicating the extent of KRAS G12C clustering, are shown in bar graphs (n = 32). Statistical significance was assessed using Student’s t-test for gold particle count (left) and bootstrap test for L max (right). Numeric values indicate P -values. Scale bar: 0.1 μm. (c) eIF2Bε depletion reduces mutant KRAS clustering. PM sheets were isolated from T47D cells stably expressing GFP-KRAS G12V or GFP-HRAS G12V along with eIF2Bε shRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized via EM. The number of gold particles is presented as mean ± SEM (n = 10). Spatial mapping was also performed, and peak L max values, reflecting the degree of protein clustering, are shown as bar graphs. Numeric values indicate P-values. ( d , e ) eIF2Bε KD significantly reduces the PM levels of GM3 and SM4. PM sheets from H358 cells (KRAS G12C; panel d) or Caco-2 cells overexpressing GFP-KRAS G12V (panel e), treated with either scrambled shRNA or eIF2Bε-targeting shRNA, were fixed and labeled with 4.5 nm gold-conjugated anti-GM3 or anti-SM4 antibodies, then imaged by EM. Spatial distribution of gold particles was analyzed using univariate K -functions (L(r) – r) . PM levels of GM3 and SM4 were quantified as gold particle density per 1 μm², and clustering was assessed by the peak value of L(r) – r ( L max ). Statistical significance for labeling density and L max was determined using Student’s t-test and bootstrap analysis, respectively (n ≥ 12, mean ± SEM). ( f ) Silencing of B4GALT5 specifically reduces GTP-bound KRAS in mutant KRAS-expressing cells. H358 (KRAS G12C) and H1703 (WT KRAS) cells were transfected with either scrambled control or B4GALT5 siRNA. Protein extracts were subjected to pull-down assays using GST–RBD of RAF, followed by immunoblotting with antibodies against KRAS, HRAS, eIF2Bε, SOS1, and B4GALT5. Protein loading was assessed by immunoblotting of whole-cell extracts (WCE).

Article Snippet: To assess ternary complex formation, 2 μg each of HIS-tagged human KRAS G12V (Acro Biosystems, Cat# KRS-H5143), Biotin-FLAG–tagged truncated SOS1 (BPS Bioscience, Cat# 100753), and recombinant tetrameric eIF2B (eIF2Bβγδε; ( )) were combined in 500 μL Lysis Buffer A. ISRIB was treated directly to a final concentration of 5 μM, with an equivalent volume of DMSO used as a negative control.

Techniques: Mutagenesis, Expressing, shRNA, Staining, Isolation, Stable Transfection, Transfection, Labeling, Control, Western Blot

Journal: PLoS ONE

Article Title: SVOP Is a Nucleotide Binding Protein

doi: 10.1371/journal.pone.0005315

Figure Lengend Snippet: Recombinant SVOP-FLAG fusion protein was purified from transfected HEK293 cells with Anti-FLAG M2 affinity gel. About 5 µg protein preparation was used in each photoaffinity labeling reaction with 100 µM 8-azido-ATP-biotin in the presence or absence of 1 mM non-photoreactive ATP. A control without UV photolysis was set up in parallel. The samples were resolved by SDS-PAGE and transferred to PVDF membrane for western blot analysis. The bound 8-azido-ATP was visualized by ExtrAvidin-HRP and anti-FLAG antibody was used to detect the proteins.

Article Snippet: 8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) (Boehringer Mannheim, Mannheim, Germany).

Techniques: Recombinant, Purification, Transfection, Labeling, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: SVOP Is a Nucleotide Binding Protein

doi: 10.1371/journal.pone.0005315

Figure Lengend Snippet: Purified SVOP-FLAG was labeled with the indicated concentrations of 8-azido-ATP-biotin and subjected to SDS-PAGE and western blot analysis. The net intensity of the regions of interest was quantified using a Kodak Image Station 440. A, Representative western blot result of SVOP-FLAG labeling as a function of 8-azido-ATP concentration. B, Quantification of the western data. The data were expressed as the intensity of 8-azido-ATP labeling normalized to SVOP protein signals. The error bars represent SEM (n = 4).

Article Snippet: 8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) (Boehringer Mannheim, Mannheim, Germany).

Techniques: Purification, Labeling, SDS Page, Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: SVOP Is a Nucleotide Binding Protein

doi: 10.1371/journal.pone.0005315

Figure Lengend Snippet: SVOP-FLAG was labeled with 100 µM 8-azido-ATP in the absence or presence of 1 mM indicated competitive nucleotides. Samples were subjected to SDS-PAGE and western blot followed by quantitative analysis. Panel A shows a representative western blot result. Panel B shows the quantification of the western blot data. The error bars represent SEM, n = 5.

Article Snippet: 8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) (Boehringer Mannheim, Mannheim, Germany).

Techniques: Labeling, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: SVOP Is a Nucleotide Binding Protein

doi: 10.1371/journal.pone.0005315

Figure Lengend Snippet: SVOP-FLAG was labeled with 100 µM 8-azido-ATP in the absence or presence of ATP (0.25–1.5 mM) or NAD (0.25–1.5 mM). Data were expressed as the percentage of 8-azido-ATP labeling according to control with no ATP or NAD in the reaction. A and B were representative western blot results of SVOP labeling in the presence of different concentration of ATP (A) or NAD (B). C shows the quantification of the western blot results. Error bars represent SEM, n = 3. The half maximum inhibition concentration of NAD and ATP on SVOP ATP binding is about 0.25 mM and 0.75 mM, respectively.

Article Snippet: 8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) (Boehringer Mannheim, Mannheim, Germany).

Techniques: Labeling, Western Blot, Concentration Assay, Inhibition, Binding Assay

Journal: PLoS ONE

Article Title: SVOP Is a Nucleotide Binding Protein

doi: 10.1371/journal.pone.0005315

Figure Lengend Snippet: A. 8-azido-ATP photoaffinity labeled SVOP-FLAG was cleaved by hydroxylamine and the samples were subjected to western blot analysis using anti-FLAG, anti-SVOP and ExtrAvidin-HRP. Arrowhead indicated an N-terminal fragment which is recognized by anti-SVOP antibody but not labeled by 8-azido-ATP. The Arrows indicate a C-terminal fragment which is labeled by 8-azido-ATP and anti-FLAG antibody. B. Only the C-terminal half of SVOP shows dominant nucleotide binding. N- and C-terminal halves of SVOP-FLAG were expressed and purified from HEK293 cells. Photoaffinity labeling was performed as described under Methods . C. Shown are the results of trypsin digestion of 8-azido-ATP labeled SVOP-FLAG. Labeled protein was digested at 37°C in the presence of trypsin. At the time periods indicated, an aliquot was withdrawn and subjected to analysis as described under Methods . The arrowheads indicate the smallest trypsinized 15 kDa fragment which is labeled by both 8-azido-ATP and anti-FLAG antibody. The proportion of total anti-FLAG and 8-azido-ATP labeling (undigested protein) was similar for the 15 KDa fragment (∼9% in both cases), consistent with it being labeled with the same efficiency as the full-length protein. Therefore the nucleotide-binding site is contained within this 15 KDa fragment. The image of 8-azido-ATP labeling blot was adjusted with contrast to show better results.

Article Snippet: 8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) (Boehringer Mannheim, Mannheim, Germany).

Techniques: Labeling, Western Blot, Binding Assay, Purification

Journal: PLoS ONE

Article Title: SVOP Is a Nucleotide Binding Protein

doi: 10.1371/journal.pone.0005315

Figure Lengend Snippet: Mutated SVOP constructs were generated by site-directed mutagenesis. Photoaffinity labeling with 8-azido-ATP was performed with the wildtype and the mutated proteins. All the mutants show similar binding capability as the wild type.

Article Snippet: 8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) (Boehringer Mannheim, Mannheim, Germany).

Techniques: Construct, Generated, Mutagenesis, Labeling, Binding Assay