Structured Review

Promega caspase 8
p38-MAPK–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities ( n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32 P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of <t>anti–caspase-8</t> (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32 P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.
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1) Product Images from "p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils"

Article Title: p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20031771

p38-MAPK–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities ( n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32 P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of anti–caspase-8 (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32 P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.
Figure Legend Snippet: p38-MAPK–dependent phosphorylations of procaspase-8 and procaspase-3 in intact cells. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were taken for Western blot analysis with an anti-phospho–p38-MAPK (P-p38) antibody. The blot shown is representative of at least eight separate experiments. (B) Alternatively, lysate samples were analyzed for IETDase (C8) and DEVDase (C3) activities ( n = 6). To adjust for differences between blood batches, the caspase activities measured after 2 h were defined as 100%, and values at other time points were compared with that level. Caspase-3 immunoprecipitates were obtained from freshly isolated or 32 P-labeled neutrophils after the indicated periods of exposure to anti-Fas Ab. (C) Unlabeled neutrophils were lysed, and the immunoprecipitates were immunoblotted with an anti–phospho-serine Ab, stripped, and reprobed with a mixture of anti–caspase-8 (detecting both the proform, pC8, and the active form) and anti–caspase-3 (detecting both the proform, pC3, and the active form) Abs and thereafter with an anti-phospho–p38-MAPK (P-p38) Ab. (D) The 32 P-labeled neutrophils were lysed, and immunoprecipitates were analyzed by gel electrophoresis and blotted. The blots were developed with a PhosphorImager and subsequently analyzed with a mixture of the anti–caspase-8 and the anti–caspase-3 Abs, stripped, and reprobed with the anti-phospho–p38-MAPK Ab. The blots and the autoradiogram in C and D are representative of at least three separate experiments.

Techniques Used: Incubation, Western Blot, Isolation, Labeling, Nucleic Acid Electrophoresis

Ser-364 and Ser-150 are conserved residues in caspases. (A) The homologous serine/threonine residue (first box) is found 11 amino acids upstream of the active site (second box) within the outlined caspases. These residues are found in the respective, large, p20 subunits of the caspases. (B) The known three dimensional structures of the p20 monomers of caspase-8 ( 34 ), caspase-3 ( 35 ), caspase-9 ( 36 ), and caspase-7 ( 37 ) are revealed with the three-dimensional structure viewer Cn3D. In these structures, the p38-MAPK putative phosphorylation site is depicted (yellow), below which is the active site of each caspase.
Figure Legend Snippet: Ser-364 and Ser-150 are conserved residues in caspases. (A) The homologous serine/threonine residue (first box) is found 11 amino acids upstream of the active site (second box) within the outlined caspases. These residues are found in the respective, large, p20 subunits of the caspases. (B) The known three dimensional structures of the p20 monomers of caspase-8 ( 34 ), caspase-3 ( 35 ), caspase-9 ( 36 ), and caspase-7 ( 37 ) are revealed with the three-dimensional structure viewer Cn3D. In these structures, the p38-MAPK putative phosphorylation site is depicted (yellow), below which is the active site of each caspase.

Techniques Used:

p38-MAPK–induced phosphorylations of caspase-8 and caspase-3 in vitro. (A and B) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ- 32 P]ATP and recombinant caspase-8 or -3 in the absence or presence of the p38-MAPK inhibitor SB203580. As controls, the same reaction was performed in the absence of caspases but in the presence of either purified proteins from E. coli transformed with an empty vector (C) or BSA. All blots were first developed with a PhosphorImager, cut, and analyzed with (A) the anti–caspase-8 (pC8 and C8) Ab or (B) the anti–caspase-3 (pC3 and C3) Ab, and, lastly, stripped and reprobed with the anti-phospho–p38-MAPK (P-p38) Ab. The illustrated autoradiograms and blots are representative of at least seven separate experiments. (C) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant caspase-8 and caspase-3 as substrates, in the presence or absence of ATP and under the same conditions as aforementioned. Thereafter, the activities of caspase-8 and caspase-3 were measured separately. The results are presented as percentage of the activities found in samples incubated in the same way but with an immunoprecipitate obtained using an isotype-matched control antibody. The data are expressed as mean ± SEM of seven separate experiments. The substrates, recombinant procaspase-8 (pC8) in D and recombinant procaspase-3 (pC3) in E, were incubated in the presence of ATP and either the immunoprecipitated active phosphorylated p38-MAPK (P-p38) or an immunoprecipitate obtained using an isotype-matched control antibody (Control). Thereafter, the in vitro amounts of the procaspases (pC8 and pC3) and caspases (C8 and C3), after incubations in the presence of (D) immunoprecipitated active Fas (FasR; n = 3) or (E) active caspase-8 ( n = 5), were analyzed by Western blotting.
Figure Legend Snippet: p38-MAPK–induced phosphorylations of caspase-8 and caspase-3 in vitro. (A and B) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ- 32 P]ATP and recombinant caspase-8 or -3 in the absence or presence of the p38-MAPK inhibitor SB203580. As controls, the same reaction was performed in the absence of caspases but in the presence of either purified proteins from E. coli transformed with an empty vector (C) or BSA. All blots were first developed with a PhosphorImager, cut, and analyzed with (A) the anti–caspase-8 (pC8 and C8) Ab or (B) the anti–caspase-3 (pC3 and C3) Ab, and, lastly, stripped and reprobed with the anti-phospho–p38-MAPK (P-p38) Ab. The illustrated autoradiograms and blots are representative of at least seven separate experiments. (C) Active phosphorylated p38-MAPK (P-p38) was immunoprecipitated and incubated with recombinant caspase-8 and caspase-3 as substrates, in the presence or absence of ATP and under the same conditions as aforementioned. Thereafter, the activities of caspase-8 and caspase-3 were measured separately. The results are presented as percentage of the activities found in samples incubated in the same way but with an immunoprecipitate obtained using an isotype-matched control antibody. The data are expressed as mean ± SEM of seven separate experiments. The substrates, recombinant procaspase-8 (pC8) in D and recombinant procaspase-3 (pC3) in E, were incubated in the presence of ATP and either the immunoprecipitated active phosphorylated p38-MAPK (P-p38) or an immunoprecipitate obtained using an isotype-matched control antibody (Control). Thereafter, the in vitro amounts of the procaspases (pC8 and pC3) and caspases (C8 and C3), after incubations in the presence of (D) immunoprecipitated active Fas (FasR; n = 3) or (E) active caspase-8 ( n = 5), were analyzed by Western blotting.

Techniques Used: In Vitro, Isolation, Incubation, Recombinant, Purification, Transformation Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ- 32 P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.
Figure Legend Snippet: Identification of phosphorylation sites on caspase-8 and caspase-3. (A) Active phosphorylated p38-MAPK immunoprecipitates from freshly isolated neutrophils were incubated with [γ- 32 P]ATP and recombinant procaspase-8 or procaspase-3. The proteins were separated by SDS–gel electrophoresis, and the separated proteins were digested in situ with trypsin. The obtained phosphopeptides were separated on cellulose TLC glass plates (elect.), followed by ascending chromatography (chrom.). The indicated electrophoresis direction is from the anode to the cathode. The plates were analyzed in a PhosphorImager as well as exposed to an X-ray film. (B) The phosphopeptides from caspase-8 or caspase-3 were eluted from the TLC plates and subjected to two-dimensional phosphoamino acid analysis. The locations of the phosphoamino acids (top) were compared with that of phosphoamino acid markers (bottom) as follows: serine (S), threonine (T), and tyrosine (Y). The phosphopeptides obtained from A were subjected to amino acid sequencing (C), and the radioactivity released in each cycle was measured by spotting onto TLC plates and exposure on a Fuji image analyzer. The phosphorylated serine residues, 364 for caspase-8 (C) and 150 for caspase-3 (C), are indicated in the sequence of the putative fragment from caspase-8 and caspase-3, respectively. The illustrated phosphomapping is representative of three experiments.

Techniques Used: Isolation, Incubation, Recombinant, SDS-Gel, Electrophoresis, In Situ, Thin Layer Chromatography, Chromatography, Phosphoamino Acid Analysis, Sequencing, Radioactivity

Caspase-8 and caspase-3 are coimmunoprecipitated with p38-MAPK. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were immunoprecipitated with an anti–p38-MAPKα or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were either analyzed with a mixture of two antibodies respectively directed against caspase-8 (both the proform, pC8, and the active form, C8) and caspase-3 (both the proform, pC3, and the active form, C3), sequentially stripped, and reprobed with antibodies against p38-MAPKα (p38α). (B) Samples were immunoprecipitated with an anti-phospho–p38-MAPK or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were sequentially analyzed with the antibodies against caspase-8, caspase-3, phospho–p38-MAPK (P-p38), p38-MAPKα (p38α), and p38-MAPKδ (p38δ). The blots in A and B are representative of at least seven separate experiments.
Figure Legend Snippet: Caspase-8 and caspase-3 are coimmunoprecipitated with p38-MAPK. Neutrophils were incubated with anti-Fas Ab for the indicated times and lysed. (A) Samples were immunoprecipitated with an anti–p38-MAPKα or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were either analyzed with a mixture of two antibodies respectively directed against caspase-8 (both the proform, pC8, and the active form, C8) and caspase-3 (both the proform, pC3, and the active form, C3), sequentially stripped, and reprobed with antibodies against p38-MAPKα (p38α). (B) Samples were immunoprecipitated with an anti-phospho–p38-MAPK or an isotype-matched control (C) antibody and subsequently assessed by Western blotting. The blots were sequentially analyzed with the antibodies against caspase-8, caspase-3, phospho–p38-MAPK (P-p38), p38-MAPKα (p38α), and p38-MAPKδ (p38δ). The blots in A and B are representative of at least seven separate experiments.

Techniques Used: Incubation, Immunoprecipitation, Western Blot

2) Product Images from "Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp."

Article Title: Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2015.00620

A virtual 2-DE gel showing the protein profile of Q. ilex mature seed embryo axis (0 h, un-imbibed) obtained by successive Pro-Q DPS and SYPRO-Ruby staining . Proteins stained with SYPRO-Ruby appear in green, while Pro-Q DPS stained proteins appear in red. The statistically significant differential phosphoprotein spots are indicated with circles for quantitative differences and with triangles for qualitative (absence/presence) differences. Numbers in red indicate the protein spots that were identified by MALDI TOF/TOF.
Figure Legend Snippet: A virtual 2-DE gel showing the protein profile of Q. ilex mature seed embryo axis (0 h, un-imbibed) obtained by successive Pro-Q DPS and SYPRO-Ruby staining . Proteins stained with SYPRO-Ruby appear in green, while Pro-Q DPS stained proteins appear in red. The statistically significant differential phosphoprotein spots are indicated with circles for quantitative differences and with triangles for qualitative (absence/presence) differences. Numbers in red indicate the protein spots that were identified by MALDI TOF/TOF.

Techniques Used: Staining

3) Product Images from "Phosphorylation-dependent stabilization of MZF1 upregulates N-cadherin expression during protein kinase CK2-mediated epithelial-mesenchymal transition"

Article Title: Phosphorylation-dependent stabilization of MZF1 upregulates N-cadherin expression during protein kinase CK2-mediated epithelial-mesenchymal transition

Journal: Oncogenesis

doi: 10.1038/s41389-018-0035-9

MZF1 is phosphorylated by protein kinase CK2. a Effects of increased CK2 activity on N-cadherin promoter activity. Promoter activities of pGL3-basic or pNcad-667 were measured in HEK293 cells expressing either pCMV-myc (E) or pCMV-myc-CK2α (C). Results of western blot analysis for the exogenous expression of myc-CK2α and in vitro kinase assays for intracellular CK2 activity are shown in the inset. GST-CS represents input GST-CS stained with Coomassie brilliant blue. 32 P-GST-CS represents phosphorylated GST-CS. Normalized luciferase activities are shown as mean ± SD for triplicate samples and are shown as fold-increase or fold-decrease relative to the activity from cells cotransfected with pNcad-667 and pCMV-myc. b CK2 phosphorylates MZF1 at serine 27. HEK293 cells were transfected with HA-MZF1 F1 (amino acid residues 1 to 240 of MZF1) or F2 (amino acid residues 120–360 of MZF1) (left) or with full-length MZF1 wt or MZF1 S27A (right). Exogenously expressed MZF1 variants were immunoprecipitated and used as substrates for in vitro kinase assays. 32 P-MZF1 represents HA-tagged MZF1 phosphorylated by CK2. c Identification of serine 27 as a CK2 phosphorylation site in MZF1 using mass spectrometry. Fragmentation spectrum (with b and y ions indicated) of a peptide spanning from amino acid 24 to 44 showing a phosphorylated serine at position 27 in MZF1. d Interaction between MZF1 and GST-tagged CK2α in vitro. Protein lysates isolated from HA-tagged MZF1-overexpressing HEK293 cells were mixed with human recombinant CK2 (GST-CK2α). Immunoprecipitation with a GST-specific Ab was followed by western blot analysis using anti-HA Ab. e Interaction between exogenous MZF1 and endogenous CK2α. HEK293 cells were transfected with Flag-MZF1, and lysates were immunoprecipitated with anti-Flag Ab (α-Flag) followed by western blot analysis using anti-CK2α Ab. Results of western blot analysis of total cell lysates are labeled as ‘Input’
Figure Legend Snippet: MZF1 is phosphorylated by protein kinase CK2. a Effects of increased CK2 activity on N-cadherin promoter activity. Promoter activities of pGL3-basic or pNcad-667 were measured in HEK293 cells expressing either pCMV-myc (E) or pCMV-myc-CK2α (C). Results of western blot analysis for the exogenous expression of myc-CK2α and in vitro kinase assays for intracellular CK2 activity are shown in the inset. GST-CS represents input GST-CS stained with Coomassie brilliant blue. 32 P-GST-CS represents phosphorylated GST-CS. Normalized luciferase activities are shown as mean ± SD for triplicate samples and are shown as fold-increase or fold-decrease relative to the activity from cells cotransfected with pNcad-667 and pCMV-myc. b CK2 phosphorylates MZF1 at serine 27. HEK293 cells were transfected with HA-MZF1 F1 (amino acid residues 1 to 240 of MZF1) or F2 (amino acid residues 120–360 of MZF1) (left) or with full-length MZF1 wt or MZF1 S27A (right). Exogenously expressed MZF1 variants were immunoprecipitated and used as substrates for in vitro kinase assays. 32 P-MZF1 represents HA-tagged MZF1 phosphorylated by CK2. c Identification of serine 27 as a CK2 phosphorylation site in MZF1 using mass spectrometry. Fragmentation spectrum (with b and y ions indicated) of a peptide spanning from amino acid 24 to 44 showing a phosphorylated serine at position 27 in MZF1. d Interaction between MZF1 and GST-tagged CK2α in vitro. Protein lysates isolated from HA-tagged MZF1-overexpressing HEK293 cells were mixed with human recombinant CK2 (GST-CK2α). Immunoprecipitation with a GST-specific Ab was followed by western blot analysis using anti-HA Ab. e Interaction between exogenous MZF1 and endogenous CK2α. HEK293 cells were transfected with Flag-MZF1, and lysates were immunoprecipitated with anti-Flag Ab (α-Flag) followed by western blot analysis using anti-CK2α Ab. Results of western blot analysis of total cell lysates are labeled as ‘Input’

Techniques Used: Activity Assay, Expressing, Western Blot, In Vitro, Staining, Luciferase, Transfection, Immunoprecipitation, Mass Spectrometry, Isolation, Recombinant, Labeling

4) Product Images from "Transgenic Production of an Anti HIV Antibody in the Barley Endosperm"

Article Title: Transgenic Production of an Anti HIV Antibody in the Barley Endosperm

Journal: PLoS ONE

doi: 10.1371/journal.pone.0140476

Detection of 2G12 antibodies in grain extracts. Grain extracts and CHO cell-derived 2G12 standards were separated by gradient SDS-PAGE under non-reducing conditions. 2G12 antibodies were detected by their binding to anti-human IgG Fcg-peroxidase. Lanes 1–3: 0.5, 1.0 and 2.0 μg of protein extracted from BG208/1E06-P4. Lanes 4–6: 0.5, 1.0 and 2.0 μg of protein extracted from BG208/1E06-P7. Lanes 7–9: 0.5, 1.0 and 2.0 μg of protein extracted from BG208/1E06-P13. Lanes 10–13: 0.5, 1.0, 2.0 and 4.0 ng 2G12 obtained from CHO cells.
Figure Legend Snippet: Detection of 2G12 antibodies in grain extracts. Grain extracts and CHO cell-derived 2G12 standards were separated by gradient SDS-PAGE under non-reducing conditions. 2G12 antibodies were detected by their binding to anti-human IgG Fcg-peroxidase. Lanes 1–3: 0.5, 1.0 and 2.0 μg of protein extracted from BG208/1E06-P4. Lanes 4–6: 0.5, 1.0 and 2.0 μg of protein extracted from BG208/1E06-P7. Lanes 7–9: 0.5, 1.0 and 2.0 μg of protein extracted from BG208/1E06-P13. Lanes 10–13: 0.5, 1.0, 2.0 and 4.0 ng 2G12 obtained from CHO cells.

Techniques Used: Derivative Assay, SDS Page, Binding Assay

Establishment of an endosperm transgene expression system. (A) Fluorescence microscopy of four grains expressing GFP driven by the oat GLO1 promoter. (B) Quantification of GFP based on Western blotting with an anti GFP antibody. (C) Total soluble protein profile obtained by Coomassie Brilliant Blue staining of 12% SDS-PAGE separations of extracts of a T 3 selection of line BG136/1E02-14-4 expressing GFP .
Figure Legend Snippet: Establishment of an endosperm transgene expression system. (A) Fluorescence microscopy of four grains expressing GFP driven by the oat GLO1 promoter. (B) Quantification of GFP based on Western blotting with an anti GFP antibody. (C) Total soluble protein profile obtained by Coomassie Brilliant Blue staining of 12% SDS-PAGE separations of extracts of a T 3 selection of line BG136/1E02-14-4 expressing GFP .

Techniques Used: Expressing, Fluorescence, Microscopy, Western Blot, Staining, SDS Page, Selection

5) Product Images from "Binding Ensemble PROfiling with (F)photoaffinity Labeling (BEProFL) Approach: Mapping the Binding Poses of HDAC8 Inhibitors"

Article Title: Binding Ensemble PROfiling with (F)photoaffinity Labeling (BEProFL) Approach: Mapping the Binding Poses of HDAC8 Inhibitors

Journal:

doi: 10.1021/jm9005077

(A) Location of the peptides (red) modified with probe 3 mapped on HDAC8 PDB:1T69 (blue model). The numbers # correspond to the entries in . The residues rendered as “sticks” correspond to the residues in the immediate proximity
Figure Legend Snippet: (A) Location of the peptides (red) modified with probe 3 mapped on HDAC8 PDB:1T69 (blue model). The numbers # correspond to the entries in . The residues rendered as “sticks” correspond to the residues in the immediate proximity

Techniques Used: Modification

MS/MS spectrum of HDAC8 tryptic peptide 222-239, GRYYSVNVPIQ D GIQDEK (see , entry 8) showing alkylation at Asp 233 . The peaks are annotated using the conventional proteomics MS/MS nomenclature (e.g., y 6 +1 ). Upper case letters denote the additional
Figure Legend Snippet: MS/MS spectrum of HDAC8 tryptic peptide 222-239, GRYYSVNVPIQ D GIQDEK (see , entry 8) showing alkylation at Asp 233 . The peaks are annotated using the conventional proteomics MS/MS nomenclature (e.g., y 6 +1 ). Upper case letters denote the additional

Techniques Used: Mass Spectrometry

MALDI-ToF MS Analysis of intact HDAC8 protein
Figure Legend Snippet: MALDI-ToF MS Analysis of intact HDAC8 protein

Techniques Used: Mass Spectrometry

Description of the grooves (G1-G3) and ridges (R1-R3) formed by the protein surface of HDAC8 (PDB:1T69).
Figure Legend Snippet: Description of the grooves (G1-G3) and ridges (R1-R3) formed by the protein surface of HDAC8 (PDB:1T69).

Techniques Used:

Characterization of biotinylated HDAC proteins and total HDAC proteins. (A) Western blot analyses of probe 2 (labeled as #) and probe 3 (labeled as *) binding to HDAC8 probed by strep-HRP and anti-HDAC8 antibodies. All the numbers stand for the concentration
Figure Legend Snippet: Characterization of biotinylated HDAC proteins and total HDAC proteins. (A) Western blot analyses of probe 2 (labeled as #) and probe 3 (labeled as *) binding to HDAC8 probed by strep-HRP and anti-HDAC8 antibodies. All the numbers stand for the concentration

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

6) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.405050

Identification of the Cys 99 –Cys 104 disulfide bond in Ero1α . A ) and this study. The cysteine residues are shown as yellow , green (outer
Figure Legend Snippet: Identification of the Cys 99 –Cys 104 disulfide bond in Ero1α . A ) and this study. The cysteine residues are shown as yellow , green (outer

Techniques Used:

The glutathione redox buffer counteracts Ero1α hyperactivity. A, where indicated, Ero1α-WT or Ero1α-C104A/C131A cells were induced with dox and treated with 1 m m BSO for 24 h. The cellular redox state of ERp57 was visualized by
Figure Legend Snippet: The glutathione redox buffer counteracts Ero1α hyperactivity. A, where indicated, Ero1α-WT or Ero1α-C104A/C131A cells were induced with dox and treated with 1 m m BSO for 24 h. The cellular redox state of ERp57 was visualized by

Techniques Used:

The regulatory Cys 94 –Cys 131 disulfide in Ero1α is destabilized when the Cys 99 –Cys 104 disulfide is absent. A , expression of His- and Myc-tagged Ero1 variants was induced with doxycycline for 24 h, and cells were NEM-treated to prevent
Figure Legend Snippet: The regulatory Cys 94 –Cys 131 disulfide in Ero1α is destabilized when the Cys 99 –Cys 104 disulfide is absent. A , expression of His- and Myc-tagged Ero1 variants was induced with doxycycline for 24 h, and cells were NEM-treated to prevent

Techniques Used: Expressing

The global transcriptional response to deregulated Ero1α activity. A , shown is a heatmap of the 159 genes found by a two-way analysis of variance to have significant changes in expression levels between non-induced (− dox ) and induced (+
Figure Legend Snippet: The global transcriptional response to deregulated Ero1α activity. A , shown is a heatmap of the 159 genes found by a two-way analysis of variance to have significant changes in expression levels between non-induced (− dox ) and induced (+

Techniques Used: Activity Assay, Expressing

Deregulation of Ero1α perturbs ER redox conditions and induces the UPR. A , where indicated, Ero1α-WT or Ero1α-C104A/C131A cells were induced with dox for 24 h and co-treated with 5 m m NAC for the last 18 h. Before lysis, cells
Figure Legend Snippet: Deregulation of Ero1α perturbs ER redox conditions and induces the UPR. A , where indicated, Ero1α-WT or Ero1α-C104A/C131A cells were induced with dox for 24 h and co-treated with 5 m m NAC for the last 18 h. Before lysis, cells

Techniques Used: Lysis

Cys104 Forms a Disulfide Bond with the Outer Active-site Cys99 in Ero1α OX2
Figure Legend Snippet: Cys104 Forms a Disulfide Bond with the Outer Active-site Cys99 in Ero1α OX2

Techniques Used:

7) Product Images from "Analysis of the Type IV Fimbrial-Subunit Gene fimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths"

Article Title: Analysis of the Type IV Fimbrial-Subunit Gene fimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.2.598-607.2001

Immunoblot analysis of whole-cell extracts of E. coli INVα and crude fimbriae of X. hyacinthi . Lane 1, molecular size markers (sizes [in kilodaltons] are indicated on the left); lane 2, E. coli INVα containing pCJOII; lane 3, E. coli INVα with plasmid pCRII; lane 4, X. hyacinthi S148 crude fimbrial preparation. For developing the immunoblot, rabbit antiserum (2 μl/ml) raised against purified fimbriae from X. hyacinthi S148 was used. The arrow indicates the 17-kDa fimbrial-subunit protein.
Figure Legend Snippet: Immunoblot analysis of whole-cell extracts of E. coli INVα and crude fimbriae of X. hyacinthi . Lane 1, molecular size markers (sizes [in kilodaltons] are indicated on the left); lane 2, E. coli INVα containing pCJOII; lane 3, E. coli INVα with plasmid pCRII; lane 4, X. hyacinthi S148 crude fimbrial preparation. For developing the immunoblot, rabbit antiserum (2 μl/ml) raised against purified fimbriae from X. hyacinthi S148 was used. The arrow indicates the 17-kDa fimbrial-subunit protein.

Techniques Used: Plasmid Preparation, Purification

8) Product Images from "Proteomics and Functional Analyses of Pepper Abscisic Acid–Responsive 1 (ABR1), Which Is Involved in Cell Death and Defense Signaling [C]), Which Is Involved in Cell Death and Defense Signaling [C] [W]"

Article Title: Proteomics and Functional Analyses of Pepper Abscisic Acid–Responsive 1 (ABR1), Which Is Involved in Cell Death and Defense Signaling [C]), Which Is Involved in Cell Death and Defense Signaling [C] [W]

Journal: The Plant Cell

doi: 10.1105/tpc.110.082081

RNA Gel Blot, 2D, and Immunoblot Analyses of the Expression of the ABR1 Protein and Gene in Pepper Leaves Infected by Xcv or Treated with ABA and SA. (A) Identification of the ABR1 protein by 1D and 2D electrophoresis and immunoblot analysis. The red circles and rectangles indicate ABR1 expression. IB, imunnoblotting. (B) Organ-specific expression of ABR1 in pepper plants. rRNA is used as loading control ( [B] to [E] ). (C) Expression of ABR1 in leaves at various times after treatment with 100 μM ABA. H, healthy leaves. (D) Expression of ABR1 and PR1 in leaves at various times after treatment with 5 mM SA. Pepper basic pathogenesis-related protein gene ( PR1 ) was used as a comparable control. H, healthy leaves. (E) Expression of the ABR1 gene in pepper leaves at various times after inoculation with the virulent (compatible) strain Ds-1 and the avirulent (incompatible) strain Bv5-4a of Xcv . H, healthy leaves. (F) Immunoblot analysis of expression of ABR1 protein in leaves at various times after inoculation with the Ds-1 and Bv5-4a strains of Xcv . Immunoblotting used a specific antiserum raised against an ABR1 peptide. H, healthy leaves; IB, imunnoblotting; CBB, Coomassie blue. [See online article for color version of this figure.]
Figure Legend Snippet: RNA Gel Blot, 2D, and Immunoblot Analyses of the Expression of the ABR1 Protein and Gene in Pepper Leaves Infected by Xcv or Treated with ABA and SA. (A) Identification of the ABR1 protein by 1D and 2D electrophoresis and immunoblot analysis. The red circles and rectangles indicate ABR1 expression. IB, imunnoblotting. (B) Organ-specific expression of ABR1 in pepper plants. rRNA is used as loading control ( [B] to [E] ). (C) Expression of ABR1 in leaves at various times after treatment with 100 μM ABA. H, healthy leaves. (D) Expression of ABR1 and PR1 in leaves at various times after treatment with 5 mM SA. Pepper basic pathogenesis-related protein gene ( PR1 ) was used as a comparable control. H, healthy leaves. (E) Expression of the ABR1 gene in pepper leaves at various times after inoculation with the virulent (compatible) strain Ds-1 and the avirulent (incompatible) strain Bv5-4a of Xcv . H, healthy leaves. (F) Immunoblot analysis of expression of ABR1 protein in leaves at various times after inoculation with the Ds-1 and Bv5-4a strains of Xcv . Immunoblotting used a specific antiserum raised against an ABR1 peptide. H, healthy leaves; IB, imunnoblotting; CBB, Coomassie blue. [See online article for color version of this figure.]

Techniques Used: Western Blot, Expressing, Infection, Two-Dimensional Gel Electrophoresis

9) Product Images from "The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis *"

Article Title: The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.006940

Loading of FadD32 reaction products onto Pks13 analyzed by SDS-PAGE. Upper , Coomassie Blue staining; lower , phosphorimaging. MW , molecular weight standards. A , loading of acyl-AMP onto Pks13. [ 14 C]C12 acid was used as a precursor. Lanes 1 , absence of
Figure Legend Snippet: Loading of FadD32 reaction products onto Pks13 analyzed by SDS-PAGE. Upper , Coomassie Blue staining; lower , phosphorimaging. MW , molecular weight standards. A , loading of acyl-AMP onto Pks13. [ 14 C]C12 acid was used as a precursor. Lanes 1 , absence of

Techniques Used: SDS Page, Staining, Molecular Weight

Loading of carboxylated chains onto Pks13. SDS-PAGE analyses. Upper , Coomassie Blue staining; lower, phosphorimaging. MW , molecular weight standards. A , loading assays of radiolabeled short chain derivatives to Pks13 wt or Pks13 αβ . Lanes
Figure Legend Snippet: Loading of carboxylated chains onto Pks13. SDS-PAGE analyses. Upper , Coomassie Blue staining; lower, phosphorimaging. MW , molecular weight standards. A , loading assays of radiolabeled short chain derivatives to Pks13 wt or Pks13 αβ . Lanes

Techniques Used: SDS Page, Staining, Molecular Weight

ESI-MS/MS analyses of the covalent binding of the carboxy-C16 chain to AT and phosphopantetheinylated C-ACP domains after tryptic digestion of Pks13. The MS/MS spectra were acquired using an LTQ-Orbitrap mass spectrometer. A , spectrum of the doubly charged
Figure Legend Snippet: ESI-MS/MS analyses of the covalent binding of the carboxy-C16 chain to AT and phosphopantetheinylated C-ACP domains after tryptic digestion of Pks13. The MS/MS spectra were acquired using an LTQ-Orbitrap mass spectrometer. A , spectrum of the doubly charged

Techniques Used: Mass Spectrometry, Binding Assay

In vitro condensation reactions in the presence of Pks13 and FadD32. The whole reaction products were deposited on silica gel and analyzed by TLC (eluent: CH 2 Cl 2 ). In both panels, lane 1 was sprayed with rhodamine and the spots were visualized by UV detection;
Figure Legend Snippet: In vitro condensation reactions in the presence of Pks13 and FadD32. The whole reaction products were deposited on silica gel and analyzed by TLC (eluent: CH 2 Cl 2 ). In both panels, lane 1 was sprayed with rhodamine and the spots were visualized by UV detection;

Techniques Used: In Vitro, Thin Layer Chromatography

Production and Purification of Recombinant M. tuberculosis Activated Pks13 Protein
Figure Legend Snippet: Production and Purification of Recombinant M. tuberculosis Activated Pks13 Protein

Techniques Used: Purification, Recombinant

Scheme of the stepwise activity of FadD32-Pks13 PKS and its domain organization. To simplify, C16 acyl chains were drawn. FadD32 synthesizes meromycoloyl-AMPs from the meromycolic acids and ATP ( 1 ). The meromycoloyl chain of these intermediates is then
Figure Legend Snippet: Scheme of the stepwise activity of FadD32-Pks13 PKS and its domain organization. To simplify, C16 acyl chains were drawn. FadD32 synthesizes meromycoloyl-AMPs from the meromycolic acids and ATP ( 1 ). The meromycoloyl chain of these intermediates is then

Techniques Used: Activity Assay

ESI-MS/MS analysis of the covalent binding of an acyl chain to the KS domain of Pks13 in the presence of FadD32, after tryptic digestion of Pks13. The spectrum was acquired using an LTQ-Orbitrap mass spectrometer. The MS/MS spectrum of the triply charged
Figure Legend Snippet: ESI-MS/MS analysis of the covalent binding of an acyl chain to the KS domain of Pks13 in the presence of FadD32, after tryptic digestion of Pks13. The spectrum was acquired using an LTQ-Orbitrap mass spectrometer. The MS/MS spectrum of the triply charged

Techniques Used: Mass Spectrometry, Binding Assay

10) Product Images from "Structural and functional insights into S-thiolation of human serum albumins"

Article Title: Structural and functional insights into S-thiolation of human serum albumins

Journal: Scientific Reports

doi: 10.1038/s41598-018-19610-9

S -Thiolation of HSA from hyperlipidemia patients. ( A ) Non-reducing SDS-PAGE ( left panel ) and native-PAGE ( right panel ) analysis of peak 1 and peak 2 HSA. ( B ) Linear mode MALDI-TOF/TOF MS spectrum of peak 1 and peak 2 HSA. ( C ) Anion-exchange liquid chromatography of DTT treated peak 1 and peak 2 HSA. Purified peak 1 and peak 2 HSA were treated with or without DTT for 3 h.
Figure Legend Snippet: S -Thiolation of HSA from hyperlipidemia patients. ( A ) Non-reducing SDS-PAGE ( left panel ) and native-PAGE ( right panel ) analysis of peak 1 and peak 2 HSA. ( B ) Linear mode MALDI-TOF/TOF MS spectrum of peak 1 and peak 2 HSA. ( C ) Anion-exchange liquid chromatography of DTT treated peak 1 and peak 2 HSA. Purified peak 1 and peak 2 HSA were treated with or without DTT for 3 h.

Techniques Used: SDS Page, Clear Native PAGE, Mass Spectrometry, Liquid Chromatography, Purification

11) Product Images from "Galactosylation and Sialylation Levels of IgG Predict Relapse in Patients With PR3-ANCA Associated Vasculitis"

Article Title: Galactosylation and Sialylation Levels of IgG Predict Relapse in Patients With PR3-ANCA Associated Vasculitis

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2017.01.033

The glycosylation profile of total IgG1 Fc at the time of an ANCA rise (T1) and at the time of a relapse (T2rel) in relapsing patients (black dots) and time-matched during remission (T2rem) in patients who remain in remission (gray dots). Dots represent individual patients, lines indicate corresponding pairs. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Wilcoxon signed rank test, p-values are shown if
Figure Legend Snippet: The glycosylation profile of total IgG1 Fc at the time of an ANCA rise (T1) and at the time of a relapse (T2rel) in relapsing patients (black dots) and time-matched during remission (T2rem) in patients who remain in remission (gray dots). Dots represent individual patients, lines indicate corresponding pairs. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Wilcoxon signed rank test, p-values are shown if

Techniques Used:

Time to relapse after an ANCA rise, (a) according to the degree of galactosylation of total IgG 1 Fc and (b) according to the degree of sialylation of total IgG 1 Fc.
Figure Legend Snippet: Time to relapse after an ANCA rise, (a) according to the degree of galactosylation of total IgG 1 Fc and (b) according to the degree of sialylation of total IgG 1 Fc.

Techniques Used:

The glycosylation profile of antigen specific PR3-ANCA IgG1 Fc at the time of an ANCA rise (T1) and at the time of a relapse (T2rel) in relapsing patients (black dots) and time-matched during remission (T2rem) in patients who remain in remission (gray dots). Dots represent individual patients, lines indicate corresponding pairs. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Wilcoxon signed rank test, p-values are shown if
Figure Legend Snippet: The glycosylation profile of antigen specific PR3-ANCA IgG1 Fc at the time of an ANCA rise (T1) and at the time of a relapse (T2rel) in relapsing patients (black dots) and time-matched during remission (T2rem) in patients who remain in remission (gray dots). Dots represent individual patients, lines indicate corresponding pairs. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Wilcoxon signed rank test, p-values are shown if

Techniques Used:

The glycosylation profile at the time of a relapse in relapsing patients and time-matched during remission in patients who remain in remission (gray dots). IgG1 Fc glycosylation of total IgG (left side, white background) and antigen specific PR3-ANCA (right side, yellow background) is shown. Dots represent individual patients. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Mann Whitney U test, p-values are shown if
Figure Legend Snippet: The glycosylation profile at the time of a relapse in relapsing patients and time-matched during remission in patients who remain in remission (gray dots). IgG1 Fc glycosylation of total IgG (left side, white background) and antigen specific PR3-ANCA (right side, yellow background) is shown. Dots represent individual patients. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Mann Whitney U test, p-values are shown if

Techniques Used: MANN-WHITNEY

LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1 ) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide. LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide.
Figure Legend Snippet: LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1 ) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide. LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide.

Techniques Used: Liquid Chromatography with Mass Spectroscopy

12) Product Images from "Camel and bovine chymosin: the relationship between their structures and cheese-making properties"

Article Title: Camel and bovine chymosin: the relationship between their structures and cheese-making properties

Journal: Acta Crystallographica Section D: Biological Crystallography

doi: 10.1107/S0907444913003260

Structures of bovine chymosin ( a ) and camel chymosin variant 2 ( b ). The active-site residues and activated water molecule are shown in red and the N-­terminal residues up to and including Tyr16 are shown in magenta. The experimentally verified glycosylation sites and N -acetylglucosamine are shown in yellow and the chloride ions are shown as green spheres; stick models are used for sulfate ions and glycerol.
Figure Legend Snippet: Structures of bovine chymosin ( a ) and camel chymosin variant 2 ( b ). The active-site residues and activated water molecule are shown in red and the N-­terminal residues up to and including Tyr16 are shown in magenta. The experimentally verified glycosylation sites and N -acetylglucosamine are shown in yellow and the chloride ions are shown as green spheres; stick models are used for sulfate ions and glycerol.

Techniques Used: Variant Assay

13) Product Images from "Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment"

Article Title: Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment

Journal: Communications Biology

doi: 10.1038/s42003-019-0334-5

HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (−) was compared with reducing OPG 22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG 22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG 22–196 and reduced carbamidomethylated OPG 22–196 were detected before and after the incubation with HtrA1 ( m / z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG ( m / z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG 22–90 , OPG 91–196 , m / z 8673 and 12205) derived from OPG 22–196 were detected 5 min after the reaction. c After the incubation of OPG 22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).
Figure Legend Snippet: HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (−) was compared with reducing OPG 22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG 22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG 22–196 and reduced carbamidomethylated OPG 22–196 were detected before and after the incubation with HtrA1 ( m / z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG ( m / z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG 22–90 , OPG 91–196 , m / z 8673 and 12205) derived from OPG 22–196 were detected 5 min after the reaction. c After the incubation of OPG 22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).

Techniques Used: Incubation, Sequencing, Software, Mass Spectrometry, Derivative Assay

14) Product Images from "Structural and functional insights into S-thiolation of human serum albumins"

Article Title: Structural and functional insights into S-thiolation of human serum albumins

Journal: Scientific Reports

doi: 10.1038/s41598-018-19610-9

S -Thiolation of oxHSA from hyperlipidemia patients. ( A ) and ( B ) Identification of S -thiolated cysteine residue of oxHSA from hyperlipidemia patient (n = 4). Number of detected samples of ( A ) S -cysteinylation and ( B ) S -homocysteinylation in each cysteine residue are shown. ( C ) and ( D ) Rasmol image of three-dimensional structure of HSA. The protein secondary structure is schematically shown and the domains are colored-coded as follows: IA, blue; IB, sky blue; IIA, green; IIB, yellow; IIIA, orange; IIIB, red. Cys90 and Cys101 are shown in a purple space-filling representation. ( E ) Graph illustrating the relationship between serum total homocysteine and HSA-bound homocysteine in normal subjects (n = 5) ( left panel ) and hyperlipidemia patients (n = 15) ( right panel ).
Figure Legend Snippet: S -Thiolation of oxHSA from hyperlipidemia patients. ( A ) and ( B ) Identification of S -thiolated cysteine residue of oxHSA from hyperlipidemia patient (n = 4). Number of detected samples of ( A ) S -cysteinylation and ( B ) S -homocysteinylation in each cysteine residue are shown. ( C ) and ( D ) Rasmol image of three-dimensional structure of HSA. The protein secondary structure is schematically shown and the domains are colored-coded as follows: IA, blue; IB, sky blue; IIA, green; IIB, yellow; IIIA, orange; IIIB, red. Cys90 and Cys101 are shown in a purple space-filling representation. ( E ) Graph illustrating the relationship between serum total homocysteine and HSA-bound homocysteine in normal subjects (n = 5) ( left panel ) and hyperlipidemia patients (n = 15) ( right panel ).

Techniques Used: IA

15) Product Images from "Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH‐dependent specificity switch of legumain"

Article Title: Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH‐dependent specificity switch of legumain

Journal: The EMBO Journal

doi: 10.15252/embj.201796750

Specificity profiling of thermolysin from Bacillus thermoproteolyticus at 75°C A The frequency distribution iceLogo for P4‐P4′ positions compared with MEROPS deposited distribution (below x ‐axis). B, C The identified cleavage sites are presented as heat maps with percent occurrence (B) and as fold‐change over the natural abundance in the human proteome (C). D Comparison of thermolysin frequency distribution plots between Aeropyrum pernix (above axis) and human (below axis) proteome. E Amino acid occurrences at P1ʹ cleavage sites.ʹ
Figure Legend Snippet: Specificity profiling of thermolysin from Bacillus thermoproteolyticus at 75°C A The frequency distribution iceLogo for P4‐P4′ positions compared with MEROPS deposited distribution (below x ‐axis). B, C The identified cleavage sites are presented as heat maps with percent occurrence (B) and as fold‐change over the natural abundance in the human proteome (C). D Comparison of thermolysin frequency distribution plots between Aeropyrum pernix (above axis) and human (below axis) proteome. E Amino acid occurrences at P1ʹ cleavage sites.ʹ

Techniques Used:

16) Product Images from "Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector"

Article Title: Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052785

Design of sDectin-1 expression vector. (A) Vector map of pDec1-nHis and pDec1-cHis, and illustration of its design to enhance sDectin-1 production. (B) Amino acid sequence of mDectin-1 from bone-marrow derived macrophage cells of C57BL/6 mice. The peptide fragment expressed in sDectin-1, the transmembrane region, the C-type lectin domain and the two potential N-linked glycosylation sites are indicated on the sequence based on alignment to protein sequence from Uniprot Accession Q6QLQ4.
Figure Legend Snippet: Design of sDectin-1 expression vector. (A) Vector map of pDec1-nHis and pDec1-cHis, and illustration of its design to enhance sDectin-1 production. (B) Amino acid sequence of mDectin-1 from bone-marrow derived macrophage cells of C57BL/6 mice. The peptide fragment expressed in sDectin-1, the transmembrane region, the C-type lectin domain and the two potential N-linked glycosylation sites are indicated on the sequence based on alignment to protein sequence from Uniprot Accession Q6QLQ4.

Techniques Used: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Mouse Assay

Binding of cHis-sDectin-1 to Saccharomyces cerevisiae cells. Purified cHis-sDectin-1 was diluted to a concentration of 25 µg/ml in blocking buffer with 500 µg/ml, 100 µg/ml or no laminarin. This was added to Saccharomyces cerevisiae yeast cells from overnight culture in blocking buffer (PBS with 3% FBS). The cells were then probed with an mDectin-1 goat polyclonal antibody (1∶200; AF1756; R D Systems) and AlexaFluor546-conjugated anti-goat antibody (1∶100; Catalog Number A-11056; Molecular Probes). After which, the cells were washed with PBS and fixed using 4% paraformaldehyde. The cells were then resuspended in PBS and visualized by phase contrast and fluorescent microscopy at (A) 200× magnification and (B) 600× magnification. Images are cropped or scaled to fit the illustration. cHis-sDectin-1 stained yeast cells was also analyzed by flow cytometry (C).
Figure Legend Snippet: Binding of cHis-sDectin-1 to Saccharomyces cerevisiae cells. Purified cHis-sDectin-1 was diluted to a concentration of 25 µg/ml in blocking buffer with 500 µg/ml, 100 µg/ml or no laminarin. This was added to Saccharomyces cerevisiae yeast cells from overnight culture in blocking buffer (PBS with 3% FBS). The cells were then probed with an mDectin-1 goat polyclonal antibody (1∶200; AF1756; R D Systems) and AlexaFluor546-conjugated anti-goat antibody (1∶100; Catalog Number A-11056; Molecular Probes). After which, the cells were washed with PBS and fixed using 4% paraformaldehyde. The cells were then resuspended in PBS and visualized by phase contrast and fluorescent microscopy at (A) 200× magnification and (B) 600× magnification. Images are cropped or scaled to fit the illustration. cHis-sDectin-1 stained yeast cells was also analyzed by flow cytometry (C).

Techniques Used: Binding Assay, Purification, Concentration Assay, Blocking Assay, Microscopy, Staining, Flow Cytometry, Cytometry

Binding of zymosan to sDectin-1. 20 or 200 ng cHis-sDectin-1 were coated on Maxisorp plates followed by FITC-zymosan. The plate was washed with PBS and imaged using a fluorescent microscope. Representative images from duplicate experiment are shown here.
Figure Legend Snippet: Binding of zymosan to sDectin-1. 20 or 200 ng cHis-sDectin-1 were coated on Maxisorp plates followed by FITC-zymosan. The plate was washed with PBS and imaged using a fluorescent microscope. Representative images from duplicate experiment are shown here.

Techniques Used: Binding Assay, Microscopy

Bioreactor fed-batch production of cHis-sDectin-1 using the cHis cell pool in 500 nM MTX. (A) Cell growth and cHis-sDectin-1 production profiles in 2 L stirred tank bioreactor. (B) Metabolite profiles of the bioreactor culture.
Figure Legend Snippet: Bioreactor fed-batch production of cHis-sDectin-1 using the cHis cell pool in 500 nM MTX. (A) Cell growth and cHis-sDectin-1 production profiles in 2 L stirred tank bioreactor. (B) Metabolite profiles of the bioreactor culture.

Techniques Used:

Structural analysis of cHis-sDectin-1. (A) Western blot analysis of untreated and PNGase F-treated cHis-sDectin-1 produced from CHO cell pool, compared to that produced by E. coli , using a horseradish peroxidase (HRP)-conjugated His-tag antibody (1∶ 2000; Catalog number 71840; Merck KGaA) (B) MALDI-TOF mass spectrometry analysis of the permethylated N-glycans released from the purified sDectin-1 produced from cHis CHO cell pool. Solid square, N-acetylglucosamine; solid circle, mannose; open circle, galactose; solid triangle, fucose; solid diamond, N-acetylneuraminic acid; open diamond, N-glycolylneuraminic acid.
Figure Legend Snippet: Structural analysis of cHis-sDectin-1. (A) Western blot analysis of untreated and PNGase F-treated cHis-sDectin-1 produced from CHO cell pool, compared to that produced by E. coli , using a horseradish peroxidase (HRP)-conjugated His-tag antibody (1∶ 2000; Catalog number 71840; Merck KGaA) (B) MALDI-TOF mass spectrometry analysis of the permethylated N-glycans released from the purified sDectin-1 produced from cHis CHO cell pool. Solid square, N-acetylglucosamine; solid circle, mannose; open circle, galactose; solid triangle, fucose; solid diamond, N-acetylneuraminic acid; open diamond, N-glycolylneuraminic acid.

Techniques Used: Western Blot, Produced, Mass Spectrometry, Purification

MTX amplification and characterization of sDectin-1 cell pools. (A) Western blotting of supernatant samples from cultures adapted to different MTX concentrations using an mDectin-1 goat polyclonal antibody (1∶1000; AF1756; R D Systems) with a HRP conjugated anti-goat antibody (1∶2000; Catalog number V8051; Promega). cHis pool and nHis pool are cell pools producing sDectin-1 with histidine tagged at the C- and N-terminals respectively. (B) Shake flask batch culture cell growth and sDectin-1 production profiles of sDectin-1 producing cell pools. Values shown represent mean values obtained from three replicate flasks. Error bars indicate the standard deviation of the experiment.
Figure Legend Snippet: MTX amplification and characterization of sDectin-1 cell pools. (A) Western blotting of supernatant samples from cultures adapted to different MTX concentrations using an mDectin-1 goat polyclonal antibody (1∶1000; AF1756; R D Systems) with a HRP conjugated anti-goat antibody (1∶2000; Catalog number V8051; Promega). cHis pool and nHis pool are cell pools producing sDectin-1 with histidine tagged at the C- and N-terminals respectively. (B) Shake flask batch culture cell growth and sDectin-1 production profiles of sDectin-1 producing cell pools. Values shown represent mean values obtained from three replicate flasks. Error bars indicate the standard deviation of the experiment.

Techniques Used: Amplification, Western Blot, Standard Deviation

Purification of cHis-sDectin-1. cHis-sDectin-1 was purified using an IMAC nickel column and buffer exchanged using a 10 kDa molecular weight cut-off ultrafiltration spin filter. The unpurified supernatant, flow-through and eluate from the IMAC column, as well as the filtrate from the spin filter were separated by SDS-PAGE and stained using (A) Coomassie and (B) silver staining.
Figure Legend Snippet: Purification of cHis-sDectin-1. cHis-sDectin-1 was purified using an IMAC nickel column and buffer exchanged using a 10 kDa molecular weight cut-off ultrafiltration spin filter. The unpurified supernatant, flow-through and eluate from the IMAC column, as well as the filtrate from the spin filter were separated by SDS-PAGE and stained using (A) Coomassie and (B) silver staining.

Techniques Used: Purification, Nickel Column, Molecular Weight, Flow Cytometry, SDS Page, Staining, Silver Staining

17) Product Images from "Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells"

Article Title: Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20101956

The RNS-modified CCL2 chemokine can be detected by specific antibodies. (A) Macrophages were cultured from the bone marrow of either wild-type (wt) or ccl2 −/− /ccr2 −/− (indicated for simplicity as ccl2 −/− ) mice. After stimulation with IFN-γ and LPS, macrophages were stained for NOS2, CCL2, or N-CCL2 (VHH-12BM). Background fluorescence with isotype-matched and secondary antibodies is reported in the graph and indicated as bkg. The graphs depict the mean fluorescence (mean ± SE, n = 10 ROIs). Statistical analysis was performed by a one-way ANOVA, followed by Tukey’s test (***, P
Figure Legend Snippet: The RNS-modified CCL2 chemokine can be detected by specific antibodies. (A) Macrophages were cultured from the bone marrow of either wild-type (wt) or ccl2 −/− /ccr2 −/− (indicated for simplicity as ccl2 −/− ) mice. After stimulation with IFN-γ and LPS, macrophages were stained for NOS2, CCL2, or N-CCL2 (VHH-12BM). Background fluorescence with isotype-matched and secondary antibodies is reported in the graph and indicated as bkg. The graphs depict the mean fluorescence (mean ± SE, n = 10 ROIs). Statistical analysis was performed by a one-way ANOVA, followed by Tukey’s test (***, P

Techniques Used: Modification, Cell Culture, Mouse Assay, Staining, Fluorescence

CCL2 nitration/nitrosylation prevents intratumoral T cell infiltration. EG7 tumor samples obtained from either wt or ccr2 −/− mice, treated or not with AT38 for 7 d (A) or MCA-203 tumor samples obtained from wt mice that had received intratumoral injections of CCL2 (0.5 µg in hydrogel; B) were stained for CD3 by immunohistochemistry. The graphs represent the quantification of immunoreactive cells (Student’s t test; ***, P
Figure Legend Snippet: CCL2 nitration/nitrosylation prevents intratumoral T cell infiltration. EG7 tumor samples obtained from either wt or ccr2 −/− mice, treated or not with AT38 for 7 d (A) or MCA-203 tumor samples obtained from wt mice that had received intratumoral injections of CCL2 (0.5 µg in hydrogel; B) were stained for CD3 by immunohistochemistry. The graphs represent the quantification of immunoreactive cells (Student’s t test; ***, P

Techniques Used: Nitration, Mouse Assay, Staining, Immunohistochemistry

RNS alter the biological activity of human and mouse CCL2. (A and B) Human or mouse CD8 + T lymphocytes (A) and human CD14 + or mouse CD11b + myeloid cells (B) were exposed to a gradient of recombinant human or mouse CCL2 (100, 10, or 1 ng/ml). Chemokines were either untreated (CCL2) or RNS treated (N-CCL2). Transmigrated cells were counted, and the results were expressed as fold induction over control. Data are representative of three different experiments and are expressed as the means ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (C and D) Fluo-4/Fura-Red–loaded human T cells (CD14 + or CD8 + ; A) or CHO cells that were or were not expressing human CCR2 (CHO or CHO-CCR2; D) were stimulated with either CCL2 or N-CCL2 and free [Ca 2+ ]i was measured by flow cytometry. Ionomycin was used as a positive control for the maximal Ca 2+ influx. Data are representative of one out of three experiments. (E) Human CD8 + and CD14 + cells were stained with either anti–human CCR2 phycoerythrin-conjugated mAb or with its isotypic control. Cell fluorescence was analyzed by flow cytometry. The mean fluorescence intensity, normalized to isotype control staining, was 13.2 ± 1.7 and 2.3 ± 0.3 for hCD14 and hCD8 cells, respectively (P ≤ 0.05). (F) Competitive binding was performed by incubating CD14 + cells or CHO-CCR2 cells with 125 I-hCCL2 in the presence of various concentrations of unlabeled, untreated (▪), or peroxynitrite-treated (Δ) hCCL2. After incubation, the cell-associated radioactivity was measured. Data are representative of three different experiments and are expressed as the means ± SE.
Figure Legend Snippet: RNS alter the biological activity of human and mouse CCL2. (A and B) Human or mouse CD8 + T lymphocytes (A) and human CD14 + or mouse CD11b + myeloid cells (B) were exposed to a gradient of recombinant human or mouse CCL2 (100, 10, or 1 ng/ml). Chemokines were either untreated (CCL2) or RNS treated (N-CCL2). Transmigrated cells were counted, and the results were expressed as fold induction over control. Data are representative of three different experiments and are expressed as the means ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (C and D) Fluo-4/Fura-Red–loaded human T cells (CD14 + or CD8 + ; A) or CHO cells that were or were not expressing human CCR2 (CHO or CHO-CCR2; D) were stimulated with either CCL2 or N-CCL2 and free [Ca 2+ ]i was measured by flow cytometry. Ionomycin was used as a positive control for the maximal Ca 2+ influx. Data are representative of one out of three experiments. (E) Human CD8 + and CD14 + cells were stained with either anti–human CCR2 phycoerythrin-conjugated mAb or with its isotypic control. Cell fluorescence was analyzed by flow cytometry. The mean fluorescence intensity, normalized to isotype control staining, was 13.2 ± 1.7 and 2.3 ± 0.3 for hCD14 and hCD8 cells, respectively (P ≤ 0.05). (F) Competitive binding was performed by incubating CD14 + cells or CHO-CCR2 cells with 125 I-hCCL2 in the presence of various concentrations of unlabeled, untreated (▪), or peroxynitrite-treated (Δ) hCCL2. After incubation, the cell-associated radioactivity was measured. Data are representative of three different experiments and are expressed as the means ± SE.

Techniques Used: Activity Assay, Recombinant, Expressing, Flow Cytometry, Cytometry, Positive Control, Staining, Fluorescence, Binding Assay, Incubation, Radioactivity

Improved intratumoral T cell migration after in vivo reduction of RNS. (A and B) Immunohistochemical staining for nitrotyrosine, NOS2, ARG1, or ARG2 (A) or CD3, CCL2, and N-CCL2 (B) in C26GM, TRAMP, and EG7 tumor samples obtained from mice either treated or not with AT38 for 7 d. The graphs represent the quantification of immunoreactive cells or areas (Student’s t test, ***, P
Figure Legend Snippet: Improved intratumoral T cell migration after in vivo reduction of RNS. (A and B) Immunohistochemical staining for nitrotyrosine, NOS2, ARG1, or ARG2 (A) or CD3, CCL2, and N-CCL2 (B) in C26GM, TRAMP, and EG7 tumor samples obtained from mice either treated or not with AT38 for 7 d. The graphs represent the quantification of immunoreactive cells or areas (Student’s t test, ***, P

Techniques Used: Migration, In Vivo, Immunohistochemistry, Staining, Mouse Assay

18) Product Images from "PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry"

Article Title: PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

Journal: Toxins

doi: 10.3390/toxins6113077

Chromatographic and electrophoretic profile of Porthidium hyoprora venom fractioning on a µ-Bondapack C18 column, monitoring elution profile at 280 nm. Emphasized in black is fraction 11 ( * ) characterized as PhTX-II PLA 2 ; Insert: Electrophoretic profile in Tricine SDS-PAGE (1) Molecular mass markers; (2) PhTX-II not reduced; (3) PhTX-II reduced with DTT (1 M).
Figure Legend Snippet: Chromatographic and electrophoretic profile of Porthidium hyoprora venom fractioning on a µ-Bondapack C18 column, monitoring elution profile at 280 nm. Emphasized in black is fraction 11 ( * ) characterized as PhTX-II PLA 2 ; Insert: Electrophoretic profile in Tricine SDS-PAGE (1) Molecular mass markers; (2) PhTX-II not reduced; (3) PhTX-II reduced with DTT (1 M).

Techniques Used: Proximity Ligation Assay, SDS Page

19) Product Images from "Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen"

Article Title: Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen

Journal: Nature communications

doi: 10.1038/ncomms3748

Modification at Ag85C residue 209 disrupts the active site structure. The catalytic triads for Ag85C-EBS (orange), Ag85C-C209S (blue) and Ag85C-DEP (green) are shown. Only the Ag85C-DEP structure possesses interactions between any of the catalytic triad. The dashed bond indicates the hydrogen bond between E228 and H260 in the Ag85C-DEP structure. Note that in the native form of the enzyme (not shown), H260 is also hydrogen bonded to S124. Most of the side chain of H260 in the Ag85C-EBS structure is disordered with only the β carbon of the side chain is observed.
Figure Legend Snippet: Modification at Ag85C residue 209 disrupts the active site structure. The catalytic triads for Ag85C-EBS (orange), Ag85C-C209S (blue) and Ag85C-DEP (green) are shown. Only the Ag85C-DEP structure possesses interactions between any of the catalytic triad. The dashed bond indicates the hydrogen bond between E228 and H260 in the Ag85C-DEP structure. Note that in the native form of the enzyme (not shown), H260 is also hydrogen bonded to S124. Most of the side chain of H260 in the Ag85C-EBS structure is disordered with only the β carbon of the side chain is observed.

Techniques Used: Modification

Inhibition of Ag85C activity and TDM/mAG production by ebselen. ( a ) Assay used to measure Ag85C enzymatic activity. The amount of resorufin butyrate converted to resorufin is monitored by measuring fluorescence at 593 nm. ( b ) Ebselen dose-dependence. Vo’/Vo represents the ratio of the initial velocities of the inhibited reaction (Vo’) and the uninhibited reaction (Vo). Error bars (representing standard deviation or SD) are calculated from triplicate reactions.
Figure Legend Snippet: Inhibition of Ag85C activity and TDM/mAG production by ebselen. ( a ) Assay used to measure Ag85C enzymatic activity. The amount of resorufin butyrate converted to resorufin is monitored by measuring fluorescence at 593 nm. ( b ) Ebselen dose-dependence. Vo’/Vo represents the ratio of the initial velocities of the inhibited reaction (Vo’) and the uninhibited reaction (Vo). Error bars (representing standard deviation or SD) are calculated from triplicate reactions.

Techniques Used: Inhibition, Activity Assay, Fluorescence, Standard Deviation

Ebselen inhibits mycolyltransfer in vitro and in Mycobacterium tuberculosis (a) The radiometric mycolyltransferase assay was used to measure the activity of the Ag85C protein in the presence of TMM, [U- 14 C] trehalose and 0 (CTL: control) or 10 µM EBS. TDM synthesis is completely abolished and the formation of TMM is reduced 70.7 % as compared to the control. ( b ) Effect of EBS treatment for 16 h at 0.5 and 1x MIC (10 and 20 µg/mL, respectively) on [ 14 C]-acetate incorporation into TMM, TDM and PE. Total lipids were extracted from bacterial cells as described 36 . The same volume of each sample was loaded per lane. The TLC was developed in the solvent system (chloroform:methanol:water; 20:4:0.5, v/v) and revealed by autoradiography. PE represents phosphatidylethanolamine. ( c ) Effect of EBS treatment for 16 h at 0.5 and 1 times the determined MIC value (10 and 20 µg/mL, respectively) on [ 14 C]-acetate incorporation into cell wall-bound mycolic acids. Cell wall-bound mycolic acid methyl esters (MAMEs) were prepared from delipidated cells 36 . The same volume of samples was loaded per lane. The TLC was developed three times in the solvent system (nhexanes: ethyl acetate; 95:5, v/v) and revealed by autoradiography. ( d ) Effect of EBS treatment for 16 h at 1x MIC (20 µg/mL) on [ 14 C]-acetate incorporation into TMM, TDM, PE and cell wall-bound mycolic acids was performed in triplicate. The amount of radioactivity incorporated in the products of interest shown in Fig. 2b and c was semiquantified using a Phosphorimager and results are expressed as the percentage of the value measured in the untreated cells. PE synthesis was used here as an internal standard to differentiate the specific inhibitory effects of EBS on TDM formation and cell wallbound mycolic acid transfer from the general and non-specific decrease in [1,2- 14 C]- acetate incorporation resulting from the loss of metabolic activity in the treated cells. ( e ) The ratio of TDM/TMM in the untreated control and samples treated with EBS. Error bars (SD) are calculated from three independently loaded thin layer chromatography experiments.
Figure Legend Snippet: Ebselen inhibits mycolyltransfer in vitro and in Mycobacterium tuberculosis (a) The radiometric mycolyltransferase assay was used to measure the activity of the Ag85C protein in the presence of TMM, [U- 14 C] trehalose and 0 (CTL: control) or 10 µM EBS. TDM synthesis is completely abolished and the formation of TMM is reduced 70.7 % as compared to the control. ( b ) Effect of EBS treatment for 16 h at 0.5 and 1x MIC (10 and 20 µg/mL, respectively) on [ 14 C]-acetate incorporation into TMM, TDM and PE. Total lipids were extracted from bacterial cells as described 36 . The same volume of each sample was loaded per lane. The TLC was developed in the solvent system (chloroform:methanol:water; 20:4:0.5, v/v) and revealed by autoradiography. PE represents phosphatidylethanolamine. ( c ) Effect of EBS treatment for 16 h at 0.5 and 1 times the determined MIC value (10 and 20 µg/mL, respectively) on [ 14 C]-acetate incorporation into cell wall-bound mycolic acids. Cell wall-bound mycolic acid methyl esters (MAMEs) were prepared from delipidated cells 36 . The same volume of samples was loaded per lane. The TLC was developed three times in the solvent system (nhexanes: ethyl acetate; 95:5, v/v) and revealed by autoradiography. ( d ) Effect of EBS treatment for 16 h at 1x MIC (20 µg/mL) on [ 14 C]-acetate incorporation into TMM, TDM, PE and cell wall-bound mycolic acids was performed in triplicate. The amount of radioactivity incorporated in the products of interest shown in Fig. 2b and c was semiquantified using a Phosphorimager and results are expressed as the percentage of the value measured in the untreated cells. PE synthesis was used here as an internal standard to differentiate the specific inhibitory effects of EBS on TDM formation and cell wallbound mycolic acid transfer from the general and non-specific decrease in [1,2- 14 C]- acetate incorporation resulting from the loss of metabolic activity in the treated cells. ( e ) The ratio of TDM/TMM in the untreated control and samples treated with EBS. Error bars (SD) are calculated from three independently loaded thin layer chromatography experiments.

Techniques Used: In Vitro, Activity Assay, CTL Assay, Thin Layer Chromatography, Autoradiography, Radioactivity

Modification of Ag85C by ebselen. ( a ) Intact mass of Ag85C with and without EBS. On the left, the deconvoluted ESI mass spectrum showing the intact mass of Ag85C. On the right, the deconvoluted ESI mass spectrum showing the intact mass of an Ag85C-EBS covalent complex. ( b ) Proposed modification of C209 by EBS. ( c ) EBS forms a reversible covalent complex with Ag85C that inhibits enzyme activity. All excess of EBS has been removed. Activity is normalized to wild type Ag85C and error bars (SD) are calculated from triplicate reactions. Bars 1 and 2 correspond to wild-type enzyme without or with EBS, respectively. Bar 3 is a sample of Ag85C-EBS covalent complex incubated with 1 mM DTT for 24 hours.
Figure Legend Snippet: Modification of Ag85C by ebselen. ( a ) Intact mass of Ag85C with and without EBS. On the left, the deconvoluted ESI mass spectrum showing the intact mass of Ag85C. On the right, the deconvoluted ESI mass spectrum showing the intact mass of an Ag85C-EBS covalent complex. ( b ) Proposed modification of C209 by EBS. ( c ) EBS forms a reversible covalent complex with Ag85C that inhibits enzyme activity. All excess of EBS has been removed. Activity is normalized to wild type Ag85C and error bars (SD) are calculated from triplicate reactions. Bars 1 and 2 correspond to wild-type enzyme without or with EBS, respectively. Bar 3 is a sample of Ag85C-EBS covalent complex incubated with 1 mM DTT for 24 hours.

Techniques Used: Modification, Activity Assay, Incubation

Potential mutations of C209 residue on the Ag85 and effect on the enzymatic activity. (a ) Sequence logo of mycobacterial Ag85. The logo is from 564 aligned Ag85 sequences. The x-axis represents the residue number based on that of Mtb Ag85C. ( b ) Mutating C209 inactivates Ag85C. Bars 1 and 2 are wild-type Ag85C without and with EBS (in excess), respectively. Bars 3–5 are single-nucleotide mutants of C209. Bar 6 is a double-nucleotide mutant of C209. Activity is normalized to wild type Ag85C and error bars (SD) are calculated from triplicate reactions.
Figure Legend Snippet: Potential mutations of C209 residue on the Ag85 and effect on the enzymatic activity. (a ) Sequence logo of mycobacterial Ag85. The logo is from 564 aligned Ag85 sequences. The x-axis represents the residue number based on that of Mtb Ag85C. ( b ) Mutating C209 inactivates Ag85C. Bars 1 and 2 are wild-type Ag85C without and with EBS (in excess), respectively. Bars 3–5 are single-nucleotide mutants of C209. Bar 6 is a double-nucleotide mutant of C209. Activity is normalized to wild type Ag85C and error bars (SD) are calculated from triplicate reactions.

Techniques Used: Activity Assay, Sequencing, Mutagenesis

Disorder near the modified C209 residue. Shown is a stereo view of the 2Fo-Fc map (blue) contoured at 1σ for the region encompassing C209 of the Ag85C-EBS structure. Bonds for all modeled atoms in the Ag85C-EBS (PDB accession code 4MQM) structure representing the loop region encompassing residues 206–235, as well as H260- S261 and 147–149 are shown and colored by CPK with carbon atom colored gold. No density representing the residues T212-N221 is observed suggesting significant flexibility. Density for the H260 side chain is incomplete suggesting moderate disorder. This contrasts with every other Ag85 structure, where the H260 conformation is well ordered by hydrogen bonding with either S124 or S148. The large U-shaped pocket immediately above C209 represents the most likely location of EBS.
Figure Legend Snippet: Disorder near the modified C209 residue. Shown is a stereo view of the 2Fo-Fc map (blue) contoured at 1σ for the region encompassing C209 of the Ag85C-EBS structure. Bonds for all modeled atoms in the Ag85C-EBS (PDB accession code 4MQM) structure representing the loop region encompassing residues 206–235, as well as H260- S261 and 147–149 are shown and colored by CPK with carbon atom colored gold. No density representing the residues T212-N221 is observed suggesting significant flexibility. Density for the H260 side chain is incomplete suggesting moderate disorder. This contrasts with every other Ag85 structure, where the H260 conformation is well ordered by hydrogen bonding with either S124 or S148. The large U-shaped pocket immediately above C209 represents the most likely location of EBS.

Techniques Used: Modification

20) Product Images from "Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution"

Article Title: Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

Journal: Analytical chemistry

doi: 10.1021/ac800080v

(a) Histogram of the distribution of the fractional labeling parameter ( θ ) determined by least-squares fitting for a set of 291 30S ribosomal peptides in a 15 N-pulse labeling experiment. (b) Histogram of the distribution of the fraction labeled amplitude parameter ( f L ) determined by least-squares fitting for a set of 405 peptides derived from a 1:3 mixture of unlabeled and “100%” 15 N-labeled 30S subunits. For both plots, a box-and-whiskers plot is shown at the top, indicating the median and the quartiles with boxes, and 1.5 times the interquartile range with whiskers. Clear outliers are indicated with open symbols at the top. For both plots, the normal distribution fitted to the entire set of values is shown as the solid line.
Figure Legend Snippet: (a) Histogram of the distribution of the fractional labeling parameter ( θ ) determined by least-squares fitting for a set of 291 30S ribosomal peptides in a 15 N-pulse labeling experiment. (b) Histogram of the distribution of the fraction labeled amplitude parameter ( f L ) determined by least-squares fitting for a set of 405 peptides derived from a 1:3 mixture of unlabeled and “100%” 15 N-labeled 30S subunits. For both plots, a box-and-whiskers plot is shown at the top, indicating the median and the quartiles with boxes, and 1.5 times the interquartile range with whiskers. Clear outliers are indicated with open symbols at the top. For both plots, the normal distribution fitted to the entire set of values is shown as the solid line.

Techniques Used: Labeling, Derivative Assay

21) Product Images from "Phosphorylation of SOS3-Like Calcium-Binding Proteins by Their Interacting SOS2-Like Protein Kinases Is a Common Regulatory Mechanism in Arabidopsis 1Phosphorylation of SOS3-Like Calcium-Binding Proteins by Their Interacting SOS2-Like Protein Kinases Is a Common Regulatory Mechanism in Arabidopsis 1 [W]Phosphorylation of SOS3-Like Calcium-Binding Proteins by Their Interacting SOS2-Like Protein Kinases Is a Common Regulatory Mechanism in Arabidopsis 1 [W] [OA]"

Article Title: Phosphorylation of SOS3-Like Calcium-Binding Proteins by Their Interacting SOS2-Like Protein Kinases Is a Common Regulatory Mechanism in Arabidopsis 1Phosphorylation of SOS3-Like Calcium-Binding Proteins by Their Interacting SOS2-Like Protein Kinases Is a Common Regulatory Mechanism in Arabidopsis 1 [W]Phosphorylation of SOS3-Like Calcium-Binding Proteins by Their Interacting SOS2-Like Protein Kinases Is a Common Regulatory Mechanism in Arabidopsis 1 [W] [OA]

Journal: Plant Physiology

doi: 10.1104/pp.111.173377

The phosphorylation site in SCaBP1 is mapped to the PFPF motif at Ser-216 by LC/MS/MS. A, Parent ion of the triply charged peptide DITTTFPSFVFHSQVEDT with one phosphate group. B, Collision-induced dissociation spectrum of DITTTFP p SFVFHSQVEDT. The major
Figure Legend Snippet: The phosphorylation site in SCaBP1 is mapped to the PFPF motif at Ser-216 by LC/MS/MS. A, Parent ion of the triply charged peptide DITTTFPSFVFHSQVEDT with one phosphate group. B, Collision-induced dissociation spectrum of DITTTFP p SFVFHSQVEDT. The major

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

The phosphorylation of SCaBP1 S216 is required for regulating AHA2 activity. SCaBP1, SCaBP1 S216A , or SCaBP1 S216D was expressed in the yeast RS-72 strain harboring two plasmids, AHA2 and PKS5. A suspension of each transformed strain was diluted in sterile
Figure Legend Snippet: The phosphorylation of SCaBP1 S216 is required for regulating AHA2 activity. SCaBP1, SCaBP1 S216A , or SCaBP1 S216D was expressed in the yeast RS-72 strain harboring two plasmids, AHA2 and PKS5. A suspension of each transformed strain was diluted in sterile

Techniques Used: Activity Assay, Transformation Assay

22) Product Images from "Proteoliposome Engineering with Cell‐Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems"

Article Title: Proteoliposome Engineering with Cell‐Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems

Journal: Advanced Science

doi: 10.1002/advs.201800524

Synthesized NHis–Cx43 was integrated into liposomal membranes and formed a hemichannel pore of NHis–Cx43. Fluorescence intensity of Cx43/DOPC liposomes containing ANTS and NHis–Cx43/10 mol% nickel‐chelating liposomes, after addition of DPX. Each symbol denotes an individual data point showing ANTS permeability ( n = 3) and the line denotes the mean. ** p
Figure Legend Snippet: Synthesized NHis–Cx43 was integrated into liposomal membranes and formed a hemichannel pore of NHis–Cx43. Fluorescence intensity of Cx43/DOPC liposomes containing ANTS and NHis–Cx43/10 mol% nickel‐chelating liposomes, after addition of DPX. Each symbol denotes an individual data point showing ANTS permeability ( n = 3) and the line denotes the mean. ** p

Techniques Used: Synthesized, Fluorescence, Permeability

23) Product Images from "Megalin Knockout Mice as an Animal Model of Low Molecular Weight Proteinuria"

Article Title: Megalin Knockout Mice as an Animal Model of Low Molecular Weight Proteinuria

Journal: The American Journal of Pathology

doi:

Urinary protein profile of wild-type and megalin −/− mice. Fifteen microliters of urine from adult ( A ) or juvenile mice ( B ) of the indicated genotypes were subjected to 4 to 15% nonreducing SDS-PAGE and staining with Coomassie Blue. The position of migration of marker proteins in the gel is shown. Protein bands that were identified by peptide sequencing are indicated. α 1 -M, α 1 -microglobulin; β 2 -M, β 2 -microglobulin; DBP, vitamin D-binding protein; F, female; Lys, lysozyme c; M, male; MUP-6, major urinary protein 6; PAP-1, pancreatitis-associated protein 1; RBP, retinol-binding protein.
Figure Legend Snippet: Urinary protein profile of wild-type and megalin −/− mice. Fifteen microliters of urine from adult ( A ) or juvenile mice ( B ) of the indicated genotypes were subjected to 4 to 15% nonreducing SDS-PAGE and staining with Coomassie Blue. The position of migration of marker proteins in the gel is shown. Protein bands that were identified by peptide sequencing are indicated. α 1 -M, α 1 -microglobulin; β 2 -M, β 2 -microglobulin; DBP, vitamin D-binding protein; F, female; Lys, lysozyme c; M, male; MUP-6, major urinary protein 6; PAP-1, pancreatitis-associated protein 1; RBP, retinol-binding protein.

Techniques Used: Mouse Assay, SDS Page, Staining, Migration, Marker, Sequencing, Binding Assay

24) Product Images from "Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo"

Article Title: Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2010.08.005

Possible origins of the unlabeled proteins from previously assembled 30S subunits appearing in the 21S assembly intermediate. (a) Schematic of the isotope-pulse experiment highlighting when the media is unlabeled and 50% 15 N-labeled, and when the 21S
Figure Legend Snippet: Possible origins of the unlabeled proteins from previously assembled 30S subunits appearing in the 21S assembly intermediate. (a) Schematic of the isotope-pulse experiment highlighting when the media is unlabeled and 50% 15 N-labeled, and when the 21S

Techniques Used: Labeling

Scaled protein levels for the 30S and 50S subunits and assembly intermediates. (a) Protein levels from a single fraction from the 30S subunit peak (blue, fraction 11) and protein levels from a single fraction from the 21S intermediate peak (orange,
Figure Legend Snippet: Scaled protein levels for the 30S and 50S subunits and assembly intermediates. (a) Protein levels from a single fraction from the 30S subunit peak (blue, fraction 11) and protein levels from a single fraction from the 21S intermediate peak (orange,

Techniques Used:

Changes in protein levels across different fractions in the sucrose gradient, and protein levels correlated with assembly maps. (a) Curves for representative proteins from the 30S subunit for each of the three defined groups, early binders (green, S15),
Figure Legend Snippet: Changes in protein levels across different fractions in the sucrose gradient, and protein levels correlated with assembly maps. (a) Curves for representative proteins from the 30S subunit for each of the three defined groups, early binders (green, S15),

Techniques Used:

25) Product Images from "Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation"

Article Title: Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000993

Galectin-1 inhibits NiV-F 0 endocytosis and maturation. A , Galectin-1 decreases internalization of NiV-F 0 from the plasma membrane. Cells transfected with NiV-F were cell surface biotinylated, then incubated in the presence of 20µM galectin-1 (bold line), or buffer control (dashed line), for the indicated times to allow internalization. Internalized biotinylated NiV-F was quantified by ELISA. Percent internalization was determined as the amount of internalized biotinylated NiV-F compared to total biotinylated NiV-F at the initial timepoint. Data are mean ± SEM for seven replicate experiments. B , Galectin-1 inhibits NiV-F 0 proteolytic processing. 293T cells expressing NiV-F were pulse-labeled with 35 S-methionine, then chased for 4 or 6 hrs in the presence or absence of galectin-1. NiV-F was immunoprecipitated with anti-NiV-F polyclonal sera and proteolytic processing analyzed by immunoblotting. C , Graphic representation of data in B. Cleavage ratio was determined as the amount of processed NiV-F (F 1 +F 2 ) compared to total NiV-F protein. Data are mean ± SEM of three replicate experiments.
Figure Legend Snippet: Galectin-1 inhibits NiV-F 0 endocytosis and maturation. A , Galectin-1 decreases internalization of NiV-F 0 from the plasma membrane. Cells transfected with NiV-F were cell surface biotinylated, then incubated in the presence of 20µM galectin-1 (bold line), or buffer control (dashed line), for the indicated times to allow internalization. Internalized biotinylated NiV-F was quantified by ELISA. Percent internalization was determined as the amount of internalized biotinylated NiV-F compared to total biotinylated NiV-F at the initial timepoint. Data are mean ± SEM for seven replicate experiments. B , Galectin-1 inhibits NiV-F 0 proteolytic processing. 293T cells expressing NiV-F were pulse-labeled with 35 S-methionine, then chased for 4 or 6 hrs in the presence or absence of galectin-1. NiV-F was immunoprecipitated with anti-NiV-F polyclonal sera and proteolytic processing analyzed by immunoblotting. C , Graphic representation of data in B. Cleavage ratio was determined as the amount of processed NiV-F (F 1 +F 2 ) compared to total NiV-F protein. Data are mean ± SEM of three replicate experiments.

Techniques Used: Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Labeling, Immunoprecipitation

Galectin-1 inhibits function of mature NiV-F. A , Chlorpromazine inhibits maturation of NiV-F 0 . PK-13 were transfected with NiV-F in the absence or presence of chlorpromazine (50µM). Cells were incubated overnight and NiV-F 0 and NiV-F 1 detected by immunoblotting. B , Galectin-1 inhibits heterologous cell fusion in the presence of chlorpromazine. BSRT7 ephrinB2 positive cells were added to a monolayer of PK13 cells transfected with NiV-F, NiV-G and a luciferase construct with a T 7 dependent promoter in the presence or absence of chlorpromazine (50µM) and galectin-1 (20µM). * p = 0.0002, calculated using Student's t test. C , Fusion kinetics in the presence or absence of galectin-1. NiV-G and NiV-F were expressed in effector PK13 cells, and the relative rate of fusion assessed on target 293T cells loaded with CCF2 dye. Relative fusion is the ratio of blue to green fluorescence from NiV-G and NiV-F-transfected effector cells minus the ratio of background blue to green fluorescence from empty-vector (pcDNA3)-transfected cells. Each data point is the mean of three independent experiments. D , Galectin-1 inhibits the ability of NiV-F to be triggered for membrane fusion. CHO cells expressing NiV-F and NiV-G were mixed with CHO cells (negative control, grey shaded) or CHOB2 cells (ephrinB2 positive, black line) for 1.5 hr at 4°C. Cell mixtures were brought to 37°C or kept at 4°C for 1.5 hr with 1 µM biotinylated HR2 peptide, in the presence or absence of galectin-1GG; top, 4°C without and with galectin-1; bottom, 37°C without and with galectin-1. E , Inhibition of F triggering at 37°C; data are mean fluorescence intensity of triplicate determinations ± SEM. See also Supplementary Figure S1 .
Figure Legend Snippet: Galectin-1 inhibits function of mature NiV-F. A , Chlorpromazine inhibits maturation of NiV-F 0 . PK-13 were transfected with NiV-F in the absence or presence of chlorpromazine (50µM). Cells were incubated overnight and NiV-F 0 and NiV-F 1 detected by immunoblotting. B , Galectin-1 inhibits heterologous cell fusion in the presence of chlorpromazine. BSRT7 ephrinB2 positive cells were added to a monolayer of PK13 cells transfected with NiV-F, NiV-G and a luciferase construct with a T 7 dependent promoter in the presence or absence of chlorpromazine (50µM) and galectin-1 (20µM). * p = 0.0002, calculated using Student's t test. C , Fusion kinetics in the presence or absence of galectin-1. NiV-G and NiV-F were expressed in effector PK13 cells, and the relative rate of fusion assessed on target 293T cells loaded with CCF2 dye. Relative fusion is the ratio of blue to green fluorescence from NiV-G and NiV-F-transfected effector cells minus the ratio of background blue to green fluorescence from empty-vector (pcDNA3)-transfected cells. Each data point is the mean of three independent experiments. D , Galectin-1 inhibits the ability of NiV-F to be triggered for membrane fusion. CHO cells expressing NiV-F and NiV-G were mixed with CHO cells (negative control, grey shaded) or CHOB2 cells (ephrinB2 positive, black line) for 1.5 hr at 4°C. Cell mixtures were brought to 37°C or kept at 4°C for 1.5 hr with 1 µM biotinylated HR2 peptide, in the presence or absence of galectin-1GG; top, 4°C without and with galectin-1; bottom, 37°C without and with galectin-1. E , Inhibition of F triggering at 37°C; data are mean fluorescence intensity of triplicate determinations ± SEM. See also Supplementary Figure S1 .

Techniques Used: Transfection, Incubation, Luciferase, Construct, Relative Rate, Fluorescence, Plasmid Preparation, Expressing, Negative Control, Inhibition

Galectin-1 blocks NiV-F and G mediated syncytia formation of endothelial and glial cells. A , Quantification of galectin-1 inhibition. PK-13 (ephrinB2 negative) cells expressing NiV-F and NiV-G were added to monolayers of ephrinB2 positive cells, Vero (control), HUVEC, mVEC, and U87. Heterologous fusion in the absence and presence of 20µM galectin-1 (white and black bars respectively) were quantified as described in Experimental Procedures. Data are mean ± SD of triplicate samples from one of three replicate experiments. B , Representative images of cell fusion in the absence or presence of galectin-1. Left panels show multinucleated syncytia in the indicated cell type. Right panels are cells treated with galectin-1 (20×).
Figure Legend Snippet: Galectin-1 blocks NiV-F and G mediated syncytia formation of endothelial and glial cells. A , Quantification of galectin-1 inhibition. PK-13 (ephrinB2 negative) cells expressing NiV-F and NiV-G were added to monolayers of ephrinB2 positive cells, Vero (control), HUVEC, mVEC, and U87. Heterologous fusion in the absence and presence of 20µM galectin-1 (white and black bars respectively) were quantified as described in Experimental Procedures. Data are mean ± SD of triplicate samples from one of three replicate experiments. B , Representative images of cell fusion in the absence or presence of galectin-1. Left panels show multinucleated syncytia in the indicated cell type. Right panels are cells treated with galectin-1 (20×).

Techniques Used: Inhibition, Expressing

Endogenous endothelial galectin-1 inhibits NiV-F and NiV-G mediated syncytia formation. A , Activated HUVECs have increased cell surface galectin-1 compared to resting cells. Flow cytometric analysis of cell surface galectin-1 on resting (light grey) and activated (dark grey) HUVECs, and on resting HUVECs plus exogenous galectin-1 (black) Data are mean ± SEM of three independent experiments, each done in triplicate. B , Activated HUVECs are resistant to NiV-F and NiV-G mediated cell fusion. Heterologous cell fusion of resting (light grey) and activated (dark grey) HUVECs, and resting HUVECs plus exogenous galectin-1 (black). Data are mean ± SD of triplicate samples from one of three replicate experiments. C , Reduction of cell surface galectin-1 by siRNA. Flow cytometric analysis of cell surface galectin-1 on resting HUVECs (grey filled), siRNA treated HUVECs (grey line), and siRNA treated HUVECS with 20µM exogenous galectin-1 (black line). D , Reduction of cell surface galectin-1 in HUVECs increases susceptibility to NiV-F and G mediated cell fusion. Heterologous cell fusion of resting cells (light grey), cells with reduced galectin-1 (white), and cells with reduced galectin-1 plus exogenous galectin-1 (dark grey). Data are mean ± SD of triplicate samples from one of three replicate experiments.
Figure Legend Snippet: Endogenous endothelial galectin-1 inhibits NiV-F and NiV-G mediated syncytia formation. A , Activated HUVECs have increased cell surface galectin-1 compared to resting cells. Flow cytometric analysis of cell surface galectin-1 on resting (light grey) and activated (dark grey) HUVECs, and on resting HUVECs plus exogenous galectin-1 (black) Data are mean ± SEM of three independent experiments, each done in triplicate. B , Activated HUVECs are resistant to NiV-F and NiV-G mediated cell fusion. Heterologous cell fusion of resting (light grey) and activated (dark grey) HUVECs, and resting HUVECs plus exogenous galectin-1 (black). Data are mean ± SD of triplicate samples from one of three replicate experiments. C , Reduction of cell surface galectin-1 by siRNA. Flow cytometric analysis of cell surface galectin-1 on resting HUVECs (grey filled), siRNA treated HUVECs (grey line), and siRNA treated HUVECS with 20µM exogenous galectin-1 (black line). D , Reduction of cell surface galectin-1 in HUVECs increases susceptibility to NiV-F and G mediated cell fusion. Heterologous cell fusion of resting cells (light grey), cells with reduced galectin-1 (white), and cells with reduced galectin-1 plus exogenous galectin-1 (dark grey). Data are mean ± SD of triplicate samples from one of three replicate experiments.

Techniques Used: Flow Cytometry

Galectin-1 interferes with lateral movement of NiV-F on the plasma membrane. A , NiV-F GFP expression on the surface of two individual Vero cells. B , NiV-F GFP promotes cell fusion when transfected into Vero cells with NiV-G. (20×) C , Galectin-1 inhibits fusion mediated by NiV-F GFP . Fusion of Vero cells in the absence (−) or presence (+) of 20µM galectin-1 was measured as in Fig. 1 . D , Galectin-1 inhibited fluorescence recovery after photobleaching. NiV-F GFP transfected Vero cells were treated with buffer control (black line with black squares) or 20µM galectin-1 (grey line with black triangles), and a portion of the membrane was bleached and measured for fluorescent recovery (y-axis) as a function of time in seconds (x-axis). Data are mean ± SD of six replicate measurements from one of two independent experiments.
Figure Legend Snippet: Galectin-1 interferes with lateral movement of NiV-F on the plasma membrane. A , NiV-F GFP expression on the surface of two individual Vero cells. B , NiV-F GFP promotes cell fusion when transfected into Vero cells with NiV-G. (20×) C , Galectin-1 inhibits fusion mediated by NiV-F GFP . Fusion of Vero cells in the absence (−) or presence (+) of 20µM galectin-1 was measured as in Fig. 1 . D , Galectin-1 inhibited fluorescence recovery after photobleaching. NiV-F GFP transfected Vero cells were treated with buffer control (black line with black squares) or 20µM galectin-1 (grey line with black triangles), and a portion of the membrane was bleached and measured for fluorescent recovery (y-axis) as a function of time in seconds (x-axis). Data are mean ± SD of six replicate measurements from one of two independent experiments.

Techniques Used: Expressing, Transfection, Fluorescence

The F3 N-glycan on NiV-F is a complex N-glycan containing putative binding sites for galectin-1. A , MALDI-TOF mass spectrum of all permethylated N-glycans from NiV-F 0 . Annotated structures were deduced by taking into account theoretical compositions and knowledge of the biosynthetic pathways (for further information, refer to http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=glyco.TOC depth=2 ). All molecular ions are [M+Na] + . Peaks labeled with * represent contaminating hexose polymers. Unlabelled peaks are non-carbohydrate contaminants or permethylation products. B , Glycan component of the F3 glycopeptide, GALEIYK N NTHDLVGDVR, and effect of sialidase S digestion. Top panel – NiV-F 0 was digested with trypsin and the peptide/glycopeptide mixture was analysed by LC-ES-MS/MS; the summed MS data for the F3 glycopeptide are shown. Bottom panel – The LC-ES-MS/MS experiment was repeated after treatment of the tryptic digest of NiV-F 0 with Sialidase S; summed MS data for the partially desialylated F3 glycopeptide are shown. Unannotated peaks correspond to peptides. Molecular ions attributable to glycopeptides are annotated with m/z values and subscripted charge states. Peaks labeled in bold correspond to molecular ions that have shifted on Sialidase S digestion; these peaks are also assigned a potential glycan structure. Symbol nomenclature is that used by the Consortium of Functional Glycomics (CFG) (see key below). See also Figure S2 . Key: Galactose (yellow circle), Mannose (green circle), GlcNAc (blue square), Fucose (red triangle), NeuAc, (purple diamond).
Figure Legend Snippet: The F3 N-glycan on NiV-F is a complex N-glycan containing putative binding sites for galectin-1. A , MALDI-TOF mass spectrum of all permethylated N-glycans from NiV-F 0 . Annotated structures were deduced by taking into account theoretical compositions and knowledge of the biosynthetic pathways (for further information, refer to http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=glyco.TOC depth=2 ). All molecular ions are [M+Na] + . Peaks labeled with * represent contaminating hexose polymers. Unlabelled peaks are non-carbohydrate contaminants or permethylation products. B , Glycan component of the F3 glycopeptide, GALEIYK N NTHDLVGDVR, and effect of sialidase S digestion. Top panel – NiV-F 0 was digested with trypsin and the peptide/glycopeptide mixture was analysed by LC-ES-MS/MS; the summed MS data for the F3 glycopeptide are shown. Bottom panel – The LC-ES-MS/MS experiment was repeated after treatment of the tryptic digest of NiV-F 0 with Sialidase S; summed MS data for the partially desialylated F3 glycopeptide are shown. Unannotated peaks correspond to peptides. Molecular ions attributable to glycopeptides are annotated with m/z values and subscripted charge states. Peaks labeled in bold correspond to molecular ions that have shifted on Sialidase S digestion; these peaks are also assigned a potential glycan structure. Symbol nomenclature is that used by the Consortium of Functional Glycomics (CFG) (see key below). See also Figure S2 . Key: Galactose (yellow circle), Mannose (green circle), GlcNAc (blue square), Fucose (red triangle), NeuAc, (purple diamond).

Techniques Used: Binding Assay, Labeling, Mass Spectrometry, Functional Assay

The F3 glycan is critical for galectin-1 inhibition of NiV-F maturation and function. A , The F3 mutant (NiV-F missing the F3 glycan) is resistant to galectin-1 inhibition of syncytia formation in endothelial and glial cells, using the heterologous cell fusion assay described in Fig. 1 . PK13 cells expressing NiV-G and either wildtype NiV-F (white) or NiV-F lacking the F3 glycan (black) were added to indicated cells in the presence of galectin-1 (HUVEC, 10µM; U87, 20µM). The y-axis shows percent inhibition of fusion. Data are mean ± SD of triplicate samples from one of three replicate experiments. * p = 0.0001, calculated using unpaired Student's t test. B , The F3 glycan on NiV-F is critical for galectin-1 inhibition of NiV-F 0 internalization. Cells transfected with NiV-F3 (lacking the F3 glycan) were cell surface biotinylated and incubated in the presence of galectin-1 (20µM) (bold line), or buffer control (dashed line), for the indicated times to allow internalization. Internalized NiV-F3 was quantified as in Fig. 4 . Data are mean ± SEM for seven replicate experiments.
Figure Legend Snippet: The F3 glycan is critical for galectin-1 inhibition of NiV-F maturation and function. A , The F3 mutant (NiV-F missing the F3 glycan) is resistant to galectin-1 inhibition of syncytia formation in endothelial and glial cells, using the heterologous cell fusion assay described in Fig. 1 . PK13 cells expressing NiV-G and either wildtype NiV-F (white) or NiV-F lacking the F3 glycan (black) were added to indicated cells in the presence of galectin-1 (HUVEC, 10µM; U87, 20µM). The y-axis shows percent inhibition of fusion. Data are mean ± SD of triplicate samples from one of three replicate experiments. * p = 0.0001, calculated using unpaired Student's t test. B , The F3 glycan on NiV-F is critical for galectin-1 inhibition of NiV-F 0 internalization. Cells transfected with NiV-F3 (lacking the F3 glycan) were cell surface biotinylated and incubated in the presence of galectin-1 (20µM) (bold line), or buffer control (dashed line), for the indicated times to allow internalization. Internalized NiV-F3 was quantified as in Fig. 4 . Data are mean ± SEM for seven replicate experiments.

Techniques Used: Inhibition, Mutagenesis, Cell Fusion Assay, Expressing, Transfection, Incubation

26) Product Images from "RPA activates the XPF‐ ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks"

Article Title: RPA activates the XPF‐ ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

Journal: The EMBO Journal

doi: 10.15252/embj.201796664

The 5′–3′ exonuclease SNM 1A can load from an incision induced by XPF ‐ ERCC 1 to digest past a crosslink (Top panel) Nuclease activity of XE on “simple fork” and +leading‐strand substrates in the presence or absence of RPA and further incubated with 0.8 nM SNM1A in a time course. (Bottom panel) Schematic representation of the nuclease assay reaction products. The blue arrow denotes incision by XE and green Pacman represents digestion by SNM1A. RPA does not affect/alter SNM1A exonuclease activity as seen in the similar stepwise digestion products of SNM1A in the presence or absence of RPA (lanes 2–9). However, the presence of a model leading strand prevents SNM1A from loading onto XE‐RPA‐induced incisions to digest the DNA substrate (lanes 10–17). (Top panel) Nuclease activity of XE‐RPA on crosslinked DNA substrates (simple fork; +leading strand) and further incubation with SNM1A in a time course. SNM1A digestion inhibition by a model nascent leading strand (as in A) is overcome when an ICL is located at the fork junction.
Figure Legend Snippet: The 5′–3′ exonuclease SNM 1A can load from an incision induced by XPF ‐ ERCC 1 to digest past a crosslink (Top panel) Nuclease activity of XE on “simple fork” and +leading‐strand substrates in the presence or absence of RPA and further incubated with 0.8 nM SNM1A in a time course. (Bottom panel) Schematic representation of the nuclease assay reaction products. The blue arrow denotes incision by XE and green Pacman represents digestion by SNM1A. RPA does not affect/alter SNM1A exonuclease activity as seen in the similar stepwise digestion products of SNM1A in the presence or absence of RPA (lanes 2–9). However, the presence of a model leading strand prevents SNM1A from loading onto XE‐RPA‐induced incisions to digest the DNA substrate (lanes 10–17). (Top panel) Nuclease activity of XE‐RPA on crosslinked DNA substrates (simple fork; +leading strand) and further incubation with SNM1A in a time course. SNM1A digestion inhibition by a model nascent leading strand (as in A) is overcome when an ICL is located at the fork junction.

Techniques Used: Activity Assay, Recombinase Polymerase Amplification, Incubation, Nuclease Assay, Inhibition

A model nascent leading strand also inhibits XE activity on a fork structure containing a single triazole interstrand crosslink ( ICL ), but the presence of RPA overcomes this inhibition Sequence and schematic structure of a “simple fork” containing a single triazole ICL at the fork junction and its predicted XE nuclease incision products when radiolabelled on the 5′‐end, based on the data obtained on non‐crosslinked fork structure in Figs 1 , 2 , 3 , 4 . Green circles denote 5′[ 32 P]‐radiolabelled nucleotides. (Top panel) Nuclease activity of XE on 3′[ 32 P]‐labelled crosslinked simple fork substrate. (Bottom panel) A schematic representation of the nuclease reaction and the incision products. (Top panel) Nuclease activity of XE on 5′[ 32 P]‐labelled model native (lanes 2 and 3) and crosslinked (lanes 4 and 5) DNA substrates. The XE incision closest to the fork junction (2 nt from the junction, 26‐mer product) is inhibited in the presence of a crosslink at the fork junction (lane 5). (Bottom panel) Schematic representation of the nuclease reaction and its incision products. Nuclease activity of XE on 3′[ 32 P]‐radiolabelled crosslinked substrate (simple fork; +leading strand) in the presence or absence of 80 nM RPA. XPF‐ERCC1 incisions reduced by a leading strand are overcome by the presence of RPA (compare lane 5 to lane 6).
Figure Legend Snippet: A model nascent leading strand also inhibits XE activity on a fork structure containing a single triazole interstrand crosslink ( ICL ), but the presence of RPA overcomes this inhibition Sequence and schematic structure of a “simple fork” containing a single triazole ICL at the fork junction and its predicted XE nuclease incision products when radiolabelled on the 5′‐end, based on the data obtained on non‐crosslinked fork structure in Figs 1 , 2 , 3 , 4 . Green circles denote 5′[ 32 P]‐radiolabelled nucleotides. (Top panel) Nuclease activity of XE on 3′[ 32 P]‐labelled crosslinked simple fork substrate. (Bottom panel) A schematic representation of the nuclease reaction and the incision products. (Top panel) Nuclease activity of XE on 5′[ 32 P]‐labelled model native (lanes 2 and 3) and crosslinked (lanes 4 and 5) DNA substrates. The XE incision closest to the fork junction (2 nt from the junction, 26‐mer product) is inhibited in the presence of a crosslink at the fork junction (lane 5). (Bottom panel) Schematic representation of the nuclease reaction and its incision products. Nuclease activity of XE on 3′[ 32 P]‐radiolabelled crosslinked substrate (simple fork; +leading strand) in the presence or absence of 80 nM RPA. XPF‐ERCC1 incisions reduced by a leading strand are overcome by the presence of RPA (compare lane 5 to lane 6).

Techniques Used: Activity Assay, Recombinase Polymerase Amplification, Inhibition, Sequencing

RPA stimulation of XPF‐ERCC1 activity on “+leading‐strand” structure is not attributed to the displacement of the model nascent leading strand or the unwinding of the fork substrates by RPA Nuclease activity of XE on the indicated fork substrates in the presence or absence of RPA. RPA specifically stimulates XE activity on a “simple fork” and “+leading‐strand” substrates. Fluorescence anisotropy assay to determine the binding constants of RPA for either “simple fork” or “+lagging‐strand” substrates. The blue diamonds denote the fluorophore‐labelled nucleotides. Error bars represent SD, n = 3. (Top panel) Outline of potential consequences of incubating “+leading‐strand” substrate radiolabelled on the model nascent leading strand with RPA, and the potential products that might be revealed by analysis on a non‐denaturing PAGE gel. (Bottom panel) Nuclease activity performed as in panel a. Reaction products were separated on a 10% non‐denaturing PAGE gel. The DNA substrates remain intact in the RPA alone reactions (lanes 4 and 9), indicating that RPA does not displace the model nascent leading strand or unwind the fork substrates, at the concentrations employed. Source data are available online for this figure.
Figure Legend Snippet: RPA stimulation of XPF‐ERCC1 activity on “+leading‐strand” structure is not attributed to the displacement of the model nascent leading strand or the unwinding of the fork substrates by RPA Nuclease activity of XE on the indicated fork substrates in the presence or absence of RPA. RPA specifically stimulates XE activity on a “simple fork” and “+leading‐strand” substrates. Fluorescence anisotropy assay to determine the binding constants of RPA for either “simple fork” or “+lagging‐strand” substrates. The blue diamonds denote the fluorophore‐labelled nucleotides. Error bars represent SD, n = 3. (Top panel) Outline of potential consequences of incubating “+leading‐strand” substrate radiolabelled on the model nascent leading strand with RPA, and the potential products that might be revealed by analysis on a non‐denaturing PAGE gel. (Bottom panel) Nuclease activity performed as in panel a. Reaction products were separated on a 10% non‐denaturing PAGE gel. The DNA substrates remain intact in the RPA alone reactions (lanes 4 and 9), indicating that RPA does not displace the model nascent leading strand or unwind the fork substrates, at the concentrations employed. Source data are available online for this figure.

Techniques Used: Recombinase Polymerase Amplification, Activity Assay, Fluorescence, Binding Assay, Polyacrylamide Gel Electrophoresis

Model for the collaborative activity of XPF ‐ ERCC 1, RPA and SNM 1A to unhook a crosslink When a single replication fork encounters an ICL, the nascent leading strand initially stalls 20–40 nt from the ICL (“−20” position; step a‐i). It gradually progresses to 1 nt from the ICL (“0” position; step a‐ii), and its arrival at the ICL triggers an XPF‐ERCC1‐RPA‐induced incision six nucleotides 5′ to the junction, in a duplex region (step a‐iii). SNM1A loads from these incisions and digests past the ICL, unhooking the ICL from the DNA duplex, leaving a residual single nucleotide moiety (step a‐iv), which has been demonstrated as the reaction product using mass spectrometry to characterise the reaction products of SNM1A activity in previous work (Wang et al , 2011 ). This enables translesion synthesis to occur and repair of the broken DNA strand via homologous recombination (step a‐v). In the event of dual replication fork convergence onto an ICL, both nascent leading strands initially stall ˜20–40 nt from the ICL (step b‐i). CMG complexes from both replication forks unload from both leading strands, as previously described (Long et al , 2014 ; Zhang et al , 2015 ) which enables one nascent leading strand to gradually progresses to 1 nt from the ICL (“0” position; step b‐ii) as previously described (Raschle et al , 2008 ; Zhang et al , 2015 ). The structure that arises at this stage is inhibitory for XPF‐ERCC1. However, in the presence of RPA, XPF‐ERCC1 will be able to incise the structure (on the lagging‐strand template associated with the fork which has progressed to 0 nt) within the duplex region, 6 nt from the ICL (step b‐iv). This XPF‐ERCC1‐RPA‐induced incision enables SNM1A to load onto and digest past the ICL (step b‐v). The net result of XPF‐ERCC1‐RPA‐SNM1A is ICL unhooking, which enable the translesion (TLS) synthesis step, where the strand extended by the TLS polymerase is the nascent leading strand which remained arrested at ˜20–40 nt from the ICL on the second converged fork and did not strike the ICL (step b‐vi). Homologous recombination‐based repair of the broken chromatid completes repair and facilitates fork restart. Data information: Black dotted arrows represent initial approach by nascent leading strands. Blue arrows represent incisions by XPF‐ERCC1; green dotted arrows represent digestion by SNM1A; maroon dotted arrows represent nascent leading strand progression.
Figure Legend Snippet: Model for the collaborative activity of XPF ‐ ERCC 1, RPA and SNM 1A to unhook a crosslink When a single replication fork encounters an ICL, the nascent leading strand initially stalls 20–40 nt from the ICL (“−20” position; step a‐i). It gradually progresses to 1 nt from the ICL (“0” position; step a‐ii), and its arrival at the ICL triggers an XPF‐ERCC1‐RPA‐induced incision six nucleotides 5′ to the junction, in a duplex region (step a‐iii). SNM1A loads from these incisions and digests past the ICL, unhooking the ICL from the DNA duplex, leaving a residual single nucleotide moiety (step a‐iv), which has been demonstrated as the reaction product using mass spectrometry to characterise the reaction products of SNM1A activity in previous work (Wang et al , 2011 ). This enables translesion synthesis to occur and repair of the broken DNA strand via homologous recombination (step a‐v). In the event of dual replication fork convergence onto an ICL, both nascent leading strands initially stall ˜20–40 nt from the ICL (step b‐i). CMG complexes from both replication forks unload from both leading strands, as previously described (Long et al , 2014 ; Zhang et al , 2015 ) which enables one nascent leading strand to gradually progresses to 1 nt from the ICL (“0” position; step b‐ii) as previously described (Raschle et al , 2008 ; Zhang et al , 2015 ). The structure that arises at this stage is inhibitory for XPF‐ERCC1. However, in the presence of RPA, XPF‐ERCC1 will be able to incise the structure (on the lagging‐strand template associated with the fork which has progressed to 0 nt) within the duplex region, 6 nt from the ICL (step b‐iv). This XPF‐ERCC1‐RPA‐induced incision enables SNM1A to load onto and digest past the ICL (step b‐v). The net result of XPF‐ERCC1‐RPA‐SNM1A is ICL unhooking, which enable the translesion (TLS) synthesis step, where the strand extended by the TLS polymerase is the nascent leading strand which remained arrested at ˜20–40 nt from the ICL on the second converged fork and did not strike the ICL (step b‐vi). Homologous recombination‐based repair of the broken chromatid completes repair and facilitates fork restart. Data information: Black dotted arrows represent initial approach by nascent leading strands. Blue arrows represent incisions by XPF‐ERCC1; green dotted arrows represent digestion by SNM1A; maroon dotted arrows represent nascent leading strand progression.

Techniques Used: Activity Assay, Recombinase Polymerase Amplification, Mass Spectrometry, Translesion Synthesis, Homologous Recombination

RPA stimulates XPF ‐ ERCC 1 activity by binding to the 5′ arms of a DNA fork substrate Fluorescence anisotropy assay to determine the binding constants of RPA for either “simple fork” or “+leading‐strand” substrates. The blue diamonds denote the fluorophore‐labelled nucleotides. Nuclease activity of XE on “+leading strand” or DNA:RNA hybrid (5′ ssRNA on the bottom strand) substrates in the presence or absence of 80 nM RPA. RPA cannot stimulate XE to overcome the inhibition of a model nascent leading strand when the 5′‐ssDNA overhang is replaced with 5′ ssRNA. Green line denotes RNA. (Top panel) Nuclease activity of XE on “+leading‐strand” substrate in the presence or absence of either the WT RPA or the truncated RPA (RPA70C442). (Bottom panel) A schematic representation of the structural domains of WT RPA and RPA70C442. Purple boxes represent the DNA‐binding domains (DBD) designated as A–F. The orange box represents the winged helix domain. RPA70, RPA32 and RPA14 denote the three subunits of RPA. Limited proteolysis assay to determine structural changes in RPA in the presence or absence of the indicated substrates. 800 nM RPA was incubated with 100 nM unlabelled DNA substrates (simple fork; +leading strand; or no DNA) prior to digestion with 500 nM trypsin in a time course. Reaction samples were separated in Bis‐Tris SDS–PAGE (4–12%) and stained with InstantBlue. Red arrows indicated tryptic digestion pattern of RPA.
Figure Legend Snippet: RPA stimulates XPF ‐ ERCC 1 activity by binding to the 5′ arms of a DNA fork substrate Fluorescence anisotropy assay to determine the binding constants of RPA for either “simple fork” or “+leading‐strand” substrates. The blue diamonds denote the fluorophore‐labelled nucleotides. Nuclease activity of XE on “+leading strand” or DNA:RNA hybrid (5′ ssRNA on the bottom strand) substrates in the presence or absence of 80 nM RPA. RPA cannot stimulate XE to overcome the inhibition of a model nascent leading strand when the 5′‐ssDNA overhang is replaced with 5′ ssRNA. Green line denotes RNA. (Top panel) Nuclease activity of XE on “+leading‐strand” substrate in the presence or absence of either the WT RPA or the truncated RPA (RPA70C442). (Bottom panel) A schematic representation of the structural domains of WT RPA and RPA70C442. Purple boxes represent the DNA‐binding domains (DBD) designated as A–F. The orange box represents the winged helix domain. RPA70, RPA32 and RPA14 denote the three subunits of RPA. Limited proteolysis assay to determine structural changes in RPA in the presence or absence of the indicated substrates. 800 nM RPA was incubated with 100 nM unlabelled DNA substrates (simple fork; +leading strand; or no DNA) prior to digestion with 500 nM trypsin in a time course. Reaction samples were separated in Bis‐Tris SDS–PAGE (4–12%) and stained with InstantBlue. Red arrows indicated tryptic digestion pattern of RPA.

Techniques Used: Recombinase Polymerase Amplification, Activity Assay, Binding Assay, Fluorescence, Inhibition, Proteolysis Assay, Incubation, SDS Page, Staining

27) Product Images from "Nanobacteria Are Mineralo Fetuin Complexes"

Article Title: Nanobacteria Are Mineralo Fetuin Complexes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0040041

Comparative SDS-PAGE and Western Blot Analysis of Nanon Proteins and Fetuin Nanon proteins and bovine fetuin resolved by 10% SDS-PAGE were visualized by silver staining (A) or transferred to nitrocellulose before probing with the mouse anti-bovine fetuin antibodies (1:10,000) (B). Lane 1, nanons (5 μg); lane 2, bovine fetuin (5 μg). Molecular weight markers (MW) are indicated on the left.
Figure Legend Snippet: Comparative SDS-PAGE and Western Blot Analysis of Nanon Proteins and Fetuin Nanon proteins and bovine fetuin resolved by 10% SDS-PAGE were visualized by silver staining (A) or transferred to nitrocellulose before probing with the mouse anti-bovine fetuin antibodies (1:10,000) (B). Lane 1, nanons (5 μg); lane 2, bovine fetuin (5 μg). Molecular weight markers (MW) are indicated on the left.

Techniques Used: SDS Page, Western Blot, Silver Staining, Molecular Weight

Silver-Stained SDS-PAGE and Western Blot Analysis of Nanon Proteins (A) Silver staining of 2.5 μg (lane 1) or 5 μg (lane 2) of nanon extract subjected to 10% SDS-PAGE. (B) Western blot performed with two distinct mouse anti-nanon antibodies (1:2,500). Lanes 3 and 5, pre-immune sera; lane 4, mice 1; lane 6, mice 2. Molecular weight markers (MW) are on the left side.
Figure Legend Snippet: Silver-Stained SDS-PAGE and Western Blot Analysis of Nanon Proteins (A) Silver staining of 2.5 μg (lane 1) or 5 μg (lane 2) of nanon extract subjected to 10% SDS-PAGE. (B) Western blot performed with two distinct mouse anti-nanon antibodies (1:2,500). Lanes 3 and 5, pre-immune sera; lane 4, mice 1; lane 6, mice 2. Molecular weight markers (MW) are on the left side.

Techniques Used: Staining, SDS Page, Western Blot, Silver Staining, Mouse Assay, Molecular Weight

SDS-PAGE and Western Blot Analysis of Proteins Extracted from Human Kidney Stones 50 μl of proteic extracts obtained from human kidney stones of 2 distinct patients were subjected to 10% SDS-PAGE and silver-stained (A). Alternatively, obtained gels were transferred on a nitrocellulose membrane before probing with anti-nanon ([B], 1:2,500) or anti-human fetuin ([C], 1:10,000) antibodies. Molecular weight markers (MW) are on the left side.
Figure Legend Snippet: SDS-PAGE and Western Blot Analysis of Proteins Extracted from Human Kidney Stones 50 μl of proteic extracts obtained from human kidney stones of 2 distinct patients were subjected to 10% SDS-PAGE and silver-stained (A). Alternatively, obtained gels were transferred on a nitrocellulose membrane before probing with anti-nanon ([B], 1:2,500) or anti-human fetuin ([C], 1:10,000) antibodies. Molecular weight markers (MW) are on the left side.

Techniques Used: SDS Page, Western Blot, Staining, Molecular Weight

28) Product Images from "Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins"

Article Title: Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins

Journal: Scientific Reports

doi: 10.1038/srep05343

Fluorescence spectra of SG free in solution and in complex with dsDNA or BDA-modified BSA. (a) Fluorescence spectra of SG in complex with dsDNA. dsDNA (1 mg/ml) was incubated with SG (100 nM) in TAE buffer for 30 min. (b) Fluorescence spectra of SG in complex with BDA-modified BSA. BSA or BDA-modified BSA (1 mg/ml) were incubated with SG (500 nM) in TAE buffer for 30 min. (c) A schematic illustration of the binding of DNA intercalators to the pyrrolated proteins.
Figure Legend Snippet: Fluorescence spectra of SG free in solution and in complex with dsDNA or BDA-modified BSA. (a) Fluorescence spectra of SG in complex with dsDNA. dsDNA (1 mg/ml) was incubated with SG (100 nM) in TAE buffer for 30 min. (b) Fluorescence spectra of SG in complex with BDA-modified BSA. BSA or BDA-modified BSA (1 mg/ml) were incubated with SG (500 nM) in TAE buffer for 30 min. (c) A schematic illustration of the binding of DNA intercalators to the pyrrolated proteins.

Techniques Used: Fluorescence, Modification, Incubation, Binding Assay

Pyrrolated proteins act as a DNA mimic. (a) Zeta potential of the BDA-treated proteins. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 10 mM sodium phosphate buffer (pH 7.4) for 24 at 37°C. (b) Temperature dependence of ionic conductivity for native and BDA-treated proteins. The BDA-modified protein was prepared by incubating 10 mg BSA with 10 mM BDA in 2.0 ml of distilled water at 37°C for 24 h. Native and BDA-treated proteins were dialyzed against distilled water, lyophilized, and subjected to the ionic conductivity measurement. (c) A schematic illustration of the electron transfer in pyrrolated proteins.
Figure Legend Snippet: Pyrrolated proteins act as a DNA mimic. (a) Zeta potential of the BDA-treated proteins. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 10 mM sodium phosphate buffer (pH 7.4) for 24 at 37°C. (b) Temperature dependence of ionic conductivity for native and BDA-treated proteins. The BDA-modified protein was prepared by incubating 10 mg BSA with 10 mM BDA in 2.0 ml of distilled water at 37°C for 24 h. Native and BDA-treated proteins were dialyzed against distilled water, lyophilized, and subjected to the ionic conductivity measurement. (c) A schematic illustration of the electron transfer in pyrrolated proteins.

Techniques Used: Activated Clotting Time Assay, Incubation, Modification

Formation of N ε -pyrrolelysine in the BDA-modified proteins. (a) HPLC detection of N ε -pyrrolelysine in the BDA-modified proteins. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. The protein samples were hydrolyzed by 2 N NaOH under argon atmosphere for 18 h at 120°C. After the alkaline hydrolysis, the samples were neutralized with hydrochloric acid and analyzed by reverse-phase HPLC. Chromatogram: upper , BDA-treated BSA; lower , BSA. (b) Chemical structure of N ε -pyrrolelysine. (c) Measurement of N ε -pyrrolelysine generated in the BDA-modified proteins. The native and modified BSAs were analyzed by LC-ESI-MS in the selected ion monitoring (SIR) mode followed by alkaline hydrolysis. (d) Binding of SG to the BDA-tretaed BSA. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 1 ml of PBS at 37°C for 24 h. (e) Changes in electric properties on the molecular surface of both sides of native and BDA-modified BSA. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. Panels : upper , native BSA; lower, BDA-treated BSA. The left and right panels represent the pair of images. The left panels shows molecules in the orientation with domain I on the left and further domains arranged counterclockwise, whereas in the right panels, domain I is on the right and the domains are arranged clockwise. Colored residues: red, negative amino acids; blue, positive amino acids; green, hydrophobic amino acids including pyrrolated lysine.
Figure Legend Snippet: Formation of N ε -pyrrolelysine in the BDA-modified proteins. (a) HPLC detection of N ε -pyrrolelysine in the BDA-modified proteins. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. The protein samples were hydrolyzed by 2 N NaOH under argon atmosphere for 18 h at 120°C. After the alkaline hydrolysis, the samples were neutralized with hydrochloric acid and analyzed by reverse-phase HPLC. Chromatogram: upper , BDA-treated BSA; lower , BSA. (b) Chemical structure of N ε -pyrrolelysine. (c) Measurement of N ε -pyrrolelysine generated in the BDA-modified proteins. The native and modified BSAs were analyzed by LC-ESI-MS in the selected ion monitoring (SIR) mode followed by alkaline hydrolysis. (d) Binding of SG to the BDA-tretaed BSA. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 1 ml of PBS at 37°C for 24 h. (e) Changes in electric properties on the molecular surface of both sides of native and BDA-modified BSA. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. Panels : upper , native BSA; lower, BDA-treated BSA. The left and right panels represent the pair of images. The left panels shows molecules in the orientation with domain I on the left and further domains arranged counterclockwise, whereas in the right panels, domain I is on the right and the domains are arranged clockwise. Colored residues: red, negative amino acids; blue, positive amino acids; green, hydrophobic amino acids including pyrrolated lysine.

Techniques Used: Modification, High Performance Liquid Chromatography, Generated, Mass Spectrometry, Binding Assay, Incubation

Pyrrolation transforms self-molecules into autoantigens. (a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.
Figure Legend Snippet: Pyrrolation transforms self-molecules into autoantigens. (a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.

Techniques Used: Mouse Assay, Modification, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

Pyrrolated proteins as a molecular target of autoimmunity. (a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left , immunoblot analysis of the modified proteins using the sera from MRL- lpr mice. Right , immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL- lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL- lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left , IgG response. Right , IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL- lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA ( left ), pyrrolated BSA ( middle ), and dsDNA ( right ) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL- lpr mice are indicated by asterisks (*, P
Figure Legend Snippet: Pyrrolated proteins as a molecular target of autoimmunity. (a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left , immunoblot analysis of the modified proteins using the sera from MRL- lpr mice. Right , immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL- lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL- lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left , IgG response. Right , IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL- lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA ( left ), pyrrolated BSA ( middle ), and dsDNA ( right ) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL- lpr mice are indicated by asterisks (*, P

Techniques Used: Modification, Mouse Assay, Enzyme-linked Immunosorbent Assay

29) Product Images from "Phosphorylation-dependent stabilization of MZF1 upregulates N-cadherin expression during protein kinase CK2-mediated epithelial-mesenchymal transition"

Article Title: Phosphorylation-dependent stabilization of MZF1 upregulates N-cadherin expression during protein kinase CK2-mediated epithelial-mesenchymal transition

Journal: Oncogenesis

doi: 10.1038/s41389-018-0035-9

MZF1 is phosphorylated by protein kinase CK2. a Effects of increased CK2 activity on N-cadherin promoter activity. Promoter activities of pGL3-basic or pNcad-667 were measured in HEK293 cells expressing either pCMV-myc (E) or pCMV-myc-CK2α (C). Results of western blot analysis for the exogenous expression of myc-CK2α and in vitro kinase assays for intracellular CK2 activity are shown in the inset. GST-CS represents input GST-CS stained with Coomassie brilliant blue. 32 P-GST-CS represents phosphorylated GST-CS. Normalized luciferase activities are shown as mean ± SD for triplicate samples and are shown as fold-increase or fold-decrease relative to the activity from cells cotransfected with pNcad-667 and pCMV-myc. b CK2 phosphorylates MZF1 at serine 27. HEK293 cells were transfected with HA-MZF1 F1 (amino acid residues 1 to 240 of MZF1) or F2 (amino acid residues 120–360 of MZF1) (left) or with full-length MZF1 wt or MZF1 S27A (right). Exogenously expressed MZF1 variants were immunoprecipitated and used as substrates for in vitro kinase assays. 32 P-MZF1 represents HA-tagged MZF1 phosphorylated by CK2. c Identification of serine 27 as a CK2 phosphorylation site in MZF1 using mass spectrometry. Fragmentation spectrum (with b and y ions indicated) of a peptide spanning from amino acid 24 to 44 showing a phosphorylated serine at position 27 in MZF1. d Interaction between MZF1 and GST-tagged CK2α in vitro. Protein lysates isolated from HA-tagged MZF1-overexpressing HEK293 cells were mixed with human recombinant CK2 (GST-CK2α). Immunoprecipitation with a GST-specific Ab was followed by western blot analysis using anti-HA Ab. e Interaction between exogenous MZF1 and endogenous CK2α. HEK293 cells were transfected with Flag-MZF1, and lysates were immunoprecipitated with anti-Flag Ab (α-Flag) followed by western blot analysis using anti-CK2α Ab. Results of western blot analysis of total cell lysates are labeled as ‘Input’
Figure Legend Snippet: MZF1 is phosphorylated by protein kinase CK2. a Effects of increased CK2 activity on N-cadherin promoter activity. Promoter activities of pGL3-basic or pNcad-667 were measured in HEK293 cells expressing either pCMV-myc (E) or pCMV-myc-CK2α (C). Results of western blot analysis for the exogenous expression of myc-CK2α and in vitro kinase assays for intracellular CK2 activity are shown in the inset. GST-CS represents input GST-CS stained with Coomassie brilliant blue. 32 P-GST-CS represents phosphorylated GST-CS. Normalized luciferase activities are shown as mean ± SD for triplicate samples and are shown as fold-increase or fold-decrease relative to the activity from cells cotransfected with pNcad-667 and pCMV-myc. b CK2 phosphorylates MZF1 at serine 27. HEK293 cells were transfected with HA-MZF1 F1 (amino acid residues 1 to 240 of MZF1) or F2 (amino acid residues 120–360 of MZF1) (left) or with full-length MZF1 wt or MZF1 S27A (right). Exogenously expressed MZF1 variants were immunoprecipitated and used as substrates for in vitro kinase assays. 32 P-MZF1 represents HA-tagged MZF1 phosphorylated by CK2. c Identification of serine 27 as a CK2 phosphorylation site in MZF1 using mass spectrometry. Fragmentation spectrum (with b and y ions indicated) of a peptide spanning from amino acid 24 to 44 showing a phosphorylated serine at position 27 in MZF1. d Interaction between MZF1 and GST-tagged CK2α in vitro. Protein lysates isolated from HA-tagged MZF1-overexpressing HEK293 cells were mixed with human recombinant CK2 (GST-CK2α). Immunoprecipitation with a GST-specific Ab was followed by western blot analysis using anti-HA Ab. e Interaction between exogenous MZF1 and endogenous CK2α. HEK293 cells were transfected with Flag-MZF1, and lysates were immunoprecipitated with anti-Flag Ab (α-Flag) followed by western blot analysis using anti-CK2α Ab. Results of western blot analysis of total cell lysates are labeled as ‘Input’

Techniques Used: Activity Assay, Expressing, Western Blot, In Vitro, Staining, Luciferase, Transfection, Immunoprecipitation, Mass Spectrometry, Isolation, Recombinant, Labeling

30) Product Images from "A single N1-methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes"

Article Title: A single N1-methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes

Journal: Scientific Reports

doi: 10.1038/s41598-018-30383-z

Loss of m 1 A 645 leads to structural changes within the LSU. ( A ) Representative gel showing structure probing with DMS or SHAPE analysis with NAI in the rrp8 G209R loss of methylation mutant. 32 P-labeled primer (helix25_StrPrb) complementary to nucleotides 2428 to 2448 of human 28S rRNA were used for the analysis. Bands corresponding to modified residues are marked in red for DMS, green for NAI and orange for both. ( B ) 2-D structure model with mapped modified residues as described in ( A ). ( C ) The residues sensitive to chemical modification are represented as spheres on a 3-D structure model of helix 25 (color scheme as in panel A). The model was made using Chimera and PDB file 4V88.
Figure Legend Snippet: Loss of m 1 A 645 leads to structural changes within the LSU. ( A ) Representative gel showing structure probing with DMS or SHAPE analysis with NAI in the rrp8 G209R loss of methylation mutant. 32 P-labeled primer (helix25_StrPrb) complementary to nucleotides 2428 to 2448 of human 28S rRNA were used for the analysis. Bands corresponding to modified residues are marked in red for DMS, green for NAI and orange for both. ( B ) 2-D structure model with mapped modified residues as described in ( A ). ( C ) The residues sensitive to chemical modification are represented as spheres on a 3-D structure model of helix 25 (color scheme as in panel A). The model was made using Chimera and PDB file 4V88.

Techniques Used: Methylation, Mutagenesis, Labeling, Modification

2D proteome analysis of rrp8 mutant. ( A ) Scanned images of a typical 2-D DIGE gel of 50 µg protein from the Cy3 labeled protein pool used for the rrp8 mutant strain (left gel: pH 4–7, right gel: pH 6–11). Differentially expressed proteins are annotated and were identified using ESI-MS/MS. Differentially expressed protein spots are annotated in red (down-regulated) or green (up-regulated). ( B ) DeCyder output showing the representative differentially expressed protein spots of Asc1, Eno2, Pgk1, Rps5, Sol3 and Rps10A along with their 3-D fluorescence intensity profiles. ( C and D ) Sol3 up-regulation is related to loss of m 1 A 645 methylation. Western blot analysis using anti-HA antibodies to analyze the expression of 6xHA tagged SOL3 in wild-type, Δ rrp8 and rrp8 G209R mutant cells. As loading control, PYK1 was detected with a specific antibody. The represented fold changes in SOL3 expression were quantified using ImageJ software ( http://imagej.nih.gov/ij/ ).
Figure Legend Snippet: 2D proteome analysis of rrp8 mutant. ( A ) Scanned images of a typical 2-D DIGE gel of 50 µg protein from the Cy3 labeled protein pool used for the rrp8 mutant strain (left gel: pH 4–7, right gel: pH 6–11). Differentially expressed proteins are annotated and were identified using ESI-MS/MS. Differentially expressed protein spots are annotated in red (down-regulated) or green (up-regulated). ( B ) DeCyder output showing the representative differentially expressed protein spots of Asc1, Eno2, Pgk1, Rps5, Sol3 and Rps10A along with their 3-D fluorescence intensity profiles. ( C and D ) Sol3 up-regulation is related to loss of m 1 A 645 methylation. Western blot analysis using anti-HA antibodies to analyze the expression of 6xHA tagged SOL3 in wild-type, Δ rrp8 and rrp8 G209R mutant cells. As loading control, PYK1 was detected with a specific antibody. The represented fold changes in SOL3 expression were quantified using ImageJ software ( http://imagej.nih.gov/ij/ ).

Techniques Used: Mutagenesis, Labeling, Mass Spectrometry, Fluorescence, Methylation, Western Blot, Expressing, Software

31) Product Images from "Galactosylation and Sialylation Levels of IgG Predict Relapse in Patients With PR3-ANCA Associated Vasculitis"

Article Title: Galactosylation and Sialylation Levels of IgG Predict Relapse in Patients With PR3-ANCA Associated Vasculitis

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2017.01.033

The glycosylation profile of antigen specific PR3-ANCA IgG1 Fc at the time of an ANCA rise (T1) and at the time of a relapse (T2rel) in relapsing patients (black dots) and time-matched during remission (T2rem) in patients who remain in remission (gray dots). Dots represent individual patients, lines indicate corresponding pairs. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Wilcoxon signed rank test, p-values are shown if
Figure Legend Snippet: The glycosylation profile of antigen specific PR3-ANCA IgG1 Fc at the time of an ANCA rise (T1) and at the time of a relapse (T2rel) in relapsing patients (black dots) and time-matched during remission (T2rem) in patients who remain in remission (gray dots). Dots represent individual patients, lines indicate corresponding pairs. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Wilcoxon signed rank test, p-values are shown if

Techniques Used:

The glycosylation profile at the time of a relapse in relapsing patients and time-matched during remission in patients who remain in remission (gray dots). IgG1 Fc glycosylation of total IgG (left side, white background) and antigen specific PR3-ANCA (right side, yellow background) is shown. Dots represent individual patients. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Mann Whitney U test, p-values are shown if
Figure Legend Snippet: The glycosylation profile at the time of a relapse in relapsing patients and time-matched during remission in patients who remain in remission (gray dots). IgG1 Fc glycosylation of total IgG (left side, white background) and antigen specific PR3-ANCA (right side, yellow background) is shown. Dots represent individual patients. The box represents the median with interquartile range, the whiskers delineate the min-max range. Significant differences were evaluated using the Mann Whitney U test, p-values are shown if

Techniques Used: MANN-WHITNEY

LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1 ) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide. LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide.
Figure Legend Snippet: LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1 ) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide. LC-MS spectra showing tryptic IgG1 glycopeptides for total IgG and PR3-ANCA belonging to an AAV patient (#9; details in Supplemental Table 1) at the time of relapse. The peaks denoted with an asterisk belong to a co-enriched contaminant (an apolipoprotein O -glycopeptide). Pep = peptide.

Techniques Used: Liquid Chromatography with Mass Spectroscopy

32) Product Images from "Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity"

Article Title: Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.004172

Western blotting on PVDF membrane probed with anti-tubulin antibody. Tubulin (0.25 mg/ml) was treated with 0 to 1500 μ m CPO. Each lane of the SDS gel was loaded with 1 μg of tubulin after the sample was reduced with DTT and denatured in the presence of SDS in a boiling water bath.
Figure Legend Snippet: Western blotting on PVDF membrane probed with anti-tubulin antibody. Tubulin (0.25 mg/ml) was treated with 0 to 1500 μ m CPO. Each lane of the SDS gel was loaded with 1 μg of tubulin after the sample was reduced with DTT and denatured in the presence of SDS in a boiling water bath.

Techniques Used: Western Blot, SDS-Gel

33) Product Images from "Positional proteomics reveals differences in N‐terminal proteoform stability"

Article Title: Positional proteomics reveals differences in N‐terminal proteoform stability

Journal: Molecular Systems Biology

doi: 10.15252/msb.20156662

GO term enrichment analysis for unstable (A) and stable (B) proteins Horizontal bar chart representations are given for significantly enriched GO terms in the human proteome ( FDR q ‐value ≤ 0.05).
Figure Legend Snippet: GO term enrichment analysis for unstable (A) and stable (B) proteins Horizontal bar chart representations are given for significantly enriched GO terms in the human proteome ( FDR q ‐value ≤ 0.05).

Techniques Used:

Experimental set‐up using pSILAC and N‐terminal COFRADIC to assess N‐terminal proteoform stability Jurkat cells were pre‐labelled with light or medium L‐Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N‐terminal COFRADIC and LC‐MS/MS analysis. Ratios between three dynamic forms of N‐terminal peptides reflect protein synthesis and degradation rates, allowing the calculation of 50% turnover times. The distribution of turnover times ( N = 1,894) calculated was not unimodal, with a sizable number of quickly turned over proteins and the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.
Figure Legend Snippet: Experimental set‐up using pSILAC and N‐terminal COFRADIC to assess N‐terminal proteoform stability Jurkat cells were pre‐labelled with light or medium L‐Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N‐terminal COFRADIC and LC‐MS/MS analysis. Ratios between three dynamic forms of N‐terminal peptides reflect protein synthesis and degradation rates, allowing the calculation of 50% turnover times. The distribution of turnover times ( N = 1,894) calculated was not unimodal, with a sizable number of quickly turned over proteins and the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.

Techniques Used: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

34) Product Images from "Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization"

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00474

Position of C39 in three-dimensional structure of recoverin bound to membrane and GRK1. (A) Topology of recoverin on membrane surface built based on NMR structures of myristoylated Ca 2+ -bound protein [PDB entry 1JSA ( Ames et al., 1997 ; Valentine et al., 2003 )]. C39 (orange), calcium ions (yellow), myristoyl residue (green) and the basic residues (K5, K11, K37, R43, and K84) in close contact with the membrane are indicated. (B) The structure of recoverin complex with GRK1 [PDB entry 2I94 ( Ames et al., 2006 )]. N-terminal amphipathic helix of GRK1 (magenta), calcium ions (yellow) and C39 (orange) are indicated. The images were created using PyMol Molecular Graphics System v.1.4.1 (Schrödinger, LLC).
Figure Legend Snippet: Position of C39 in three-dimensional structure of recoverin bound to membrane and GRK1. (A) Topology of recoverin on membrane surface built based on NMR structures of myristoylated Ca 2+ -bound protein [PDB entry 1JSA ( Ames et al., 1997 ; Valentine et al., 2003 )]. C39 (orange), calcium ions (yellow), myristoyl residue (green) and the basic residues (K5, K11, K37, R43, and K84) in close contact with the membrane are indicated. (B) The structure of recoverin complex with GRK1 [PDB entry 2I94 ( Ames et al., 2006 )]. N-terminal amphipathic helix of GRK1 (magenta), calcium ions (yellow) and C39 (orange) are indicated. The images were created using PyMol Molecular Graphics System v.1.4.1 (Schrödinger, LLC).

Techniques Used: Nuclear Magnetic Resonance

Hypothetical scheme describing potential roles of disulfide dimer and thiol-oxidized monomer of recoverin in the mechanisms of photoreceptor apoptosis induced by visual light. The designations are as follows: Arr, arrestin; dArr, disulfide dimer of arrestin; GRK1, G-protein coupled kinase-1; Gt, transducin; PDE6, rod cGMP-specific 3’,5’-cyclic phosphodiesterase; Rho, rhodopsin; p Rho, phosphorylated rhodopsin; OmRec, monomeric recoverin with C39 converted into sulfinic acid; dRec, disulfide dimer of recoverin; JNK3, c-Jun N-terminal kinase 3; c-Jun/c-Fos (AP-1), activator protein 1 – heterodimeric transcription factor; nNOS, neuronal nitric oxide synthase 1; GC, guanylate cyclase; MDM2, mouse double minute 2 homolog – E3 ubiquitin-protein ligase; UPR, unfolded protein response; CHOP, C/EBP homologous protein – pro-apoptotic transcription factor; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase – translation initiation factor 2-alpha kinase 3; p eLF2α, phosphorylated translation initiation factor 2α; ATF4, activating transcription factor 4; Bcl2, B-cell lymphoma 2 – apoptosis regulator protein. For details, refer to section “Discussion.”
Figure Legend Snippet: Hypothetical scheme describing potential roles of disulfide dimer and thiol-oxidized monomer of recoverin in the mechanisms of photoreceptor apoptosis induced by visual light. The designations are as follows: Arr, arrestin; dArr, disulfide dimer of arrestin; GRK1, G-protein coupled kinase-1; Gt, transducin; PDE6, rod cGMP-specific 3’,5’-cyclic phosphodiesterase; Rho, rhodopsin; p Rho, phosphorylated rhodopsin; OmRec, monomeric recoverin with C39 converted into sulfinic acid; dRec, disulfide dimer of recoverin; JNK3, c-Jun N-terminal kinase 3; c-Jun/c-Fos (AP-1), activator protein 1 – heterodimeric transcription factor; nNOS, neuronal nitric oxide synthase 1; GC, guanylate cyclase; MDM2, mouse double minute 2 homolog – E3 ubiquitin-protein ligase; UPR, unfolded protein response; CHOP, C/EBP homologous protein – pro-apoptotic transcription factor; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase – translation initiation factor 2-alpha kinase 3; p eLF2α, phosphorylated translation initiation factor 2α; ATF4, activating transcription factor 4; Bcl2, B-cell lymphoma 2 – apoptosis regulator protein. For details, refer to section “Discussion.”

Techniques Used:

Identification of monomeric oxidized forms of recoverin in the retina of pigmented rabbits exposed to different doses of visual light illumination. MALDI-TOF/TOF mass spectra of monomeric recoverin ([MH] + molecular ions of full-length proteins) extracted from the rabbit retinas illuminated in vivo for 3 h with halogen lamp following scheme 1 (2,200 lx) (A) or scheme 2 (30,000 lx) (B) .
Figure Legend Snippet: Identification of monomeric oxidized forms of recoverin in the retina of pigmented rabbits exposed to different doses of visual light illumination. MALDI-TOF/TOF mass spectra of monomeric recoverin ([MH] + molecular ions of full-length proteins) extracted from the rabbit retinas illuminated in vivo for 3 h with halogen lamp following scheme 1 (2,200 lx) (A) or scheme 2 (30,000 lx) (B) .

Techniques Used: In Vivo

Structural properties of recoverin forms. (A,B) Far-UV CD spectra of Ca 2+ -free (A) or Ca 2+ -loaded (B) RmRec (4 μM, solid curves), OmRec (4 μM, dotted curves) and dRec (2 μM, thick solid curves) at 20°C, pH 8.2. (C) Thermal dependencies of fluorescence spectrum maximum position for Ca 2+ -free (open symbols) and Ca 2+ -loaded (filled symbols) RmRec (14 μM, squares), OmRec (14 μM, circles) and dRec (7 μM, triangles) samples at pH 7.3. (D) The binding of bis-ANS (1 μM) to Ca 2+ -free (dashed curves) or Ca 2+ -loaded (solid curves) RmRec (6 μM, medium curves), C39D mutant (6 μM, thin curves) and dRec (3 μM, thick curves) monitored by fluorescence emission spectrum of the dye at 20°C, pH 7.3.
Figure Legend Snippet: Structural properties of recoverin forms. (A,B) Far-UV CD spectra of Ca 2+ -free (A) or Ca 2+ -loaded (B) RmRec (4 μM, solid curves), OmRec (4 μM, dotted curves) and dRec (2 μM, thick solid curves) at 20°C, pH 8.2. (C) Thermal dependencies of fluorescence spectrum maximum position for Ca 2+ -free (open symbols) and Ca 2+ -loaded (filled symbols) RmRec (14 μM, squares), OmRec (14 μM, circles) and dRec (7 μM, triangles) samples at pH 7.3. (D) The binding of bis-ANS (1 μM) to Ca 2+ -free (dashed curves) or Ca 2+ -loaded (solid curves) RmRec (6 μM, medium curves), C39D mutant (6 μM, thin curves) and dRec (3 μM, thick curves) monitored by fluorescence emission spectrum of the dye at 20°C, pH 7.3.

Techniques Used: Fluorescence, Binding Assay, Mutagenesis

Identification of multimeric oxidized forms of recoverin in the retina of albino rats and pigmented rabbits exposed to different doses of visual light illumination. Western blotting under non-reducing or reducing conditions of recoverin fractions extracted from the rat retinas illuminated in vivo for 14 h with metal halide lamp (2,500 lx, ‘exp’) (A) , and rabbit retinas illuminated in vivo for 3 h with halogen lamp following scheme 1 (2,200 lx, ‘exp S1’) or scheme 2 (30,000 lx, ‘exp S2’) (B) . The recoverin fractions obtained from the dark-adapted retinas were used as a control (‘contr’). ‘M,’ ‘M2,’ and ‘A’ denote monomeric, dimeric, and multimeric/aggregated forms of recoverin, respectively. Volumes of the loaded recoverin samples were chosen to ensure nearly equivalent bands of the monomeric protein. The numbers in left-hand columns indicate the molecular masses of protein markers in kDa. (C) The weight fractions of monomeric and dimeric forms of rabbit recoverin estimated from the Western blotting data from at least three independent in vivo experiments.
Figure Legend Snippet: Identification of multimeric oxidized forms of recoverin in the retina of albino rats and pigmented rabbits exposed to different doses of visual light illumination. Western blotting under non-reducing or reducing conditions of recoverin fractions extracted from the rat retinas illuminated in vivo for 14 h with metal halide lamp (2,500 lx, ‘exp’) (A) , and rabbit retinas illuminated in vivo for 3 h with halogen lamp following scheme 1 (2,200 lx, ‘exp S1’) or scheme 2 (30,000 lx, ‘exp S2’) (B) . The recoverin fractions obtained from the dark-adapted retinas were used as a control (‘contr’). ‘M,’ ‘M2,’ and ‘A’ denote monomeric, dimeric, and multimeric/aggregated forms of recoverin, respectively. Volumes of the loaded recoverin samples were chosen to ensure nearly equivalent bands of the monomeric protein. The numbers in left-hand columns indicate the molecular masses of protein markers in kDa. (C) The weight fractions of monomeric and dimeric forms of rabbit recoverin estimated from the Western blotting data from at least three independent in vivo experiments.

Techniques Used: Western Blot, In Vivo

Functional properties of the recoverin forms. (A) The binding of 30 μM RmRec (open circles) and 15 μM dRec (open squares) to photoreceptor membranes. Recoverin was mixed with urea-washed photoreceptor membranes in the presence of 0.11–500 μM [Ca 2+ ] free at 37°C, pH 8.0 and the membranes were separated by ultracentrifugation. The fractions of membrane-bound protein evaluated by SDS-PAGE were plotted versus [Ca 2+ ] free and the plots were fitted to the 4-parameter Hill equation. Solid and dashed curves represent best fits for RmRec and dRec, respectively. The inset: fractions of the Ca 2+ -free and Ca 2+ -saturated protein bound to the membranes. (B) Inhibition of GRK1 by RmRec or C39D mutant (40 μM), or dRec (20 μM). Rhodopsin phosphorylation by GRK1 in the presence of [γ - 32 P]ATP was monitored at high [Ca 2+ ] free (200 μM Ca 2+ , filled bars) or low [Ca 2+ ] free (0.01 μM, open bars) by phosphorimaging radioautography.
Figure Legend Snippet: Functional properties of the recoverin forms. (A) The binding of 30 μM RmRec (open circles) and 15 μM dRec (open squares) to photoreceptor membranes. Recoverin was mixed with urea-washed photoreceptor membranes in the presence of 0.11–500 μM [Ca 2+ ] free at 37°C, pH 8.0 and the membranes were separated by ultracentrifugation. The fractions of membrane-bound protein evaluated by SDS-PAGE were plotted versus [Ca 2+ ] free and the plots were fitted to the 4-parameter Hill equation. Solid and dashed curves represent best fits for RmRec and dRec, respectively. The inset: fractions of the Ca 2+ -free and Ca 2+ -saturated protein bound to the membranes. (B) Inhibition of GRK1 by RmRec or C39D mutant (40 μM), or dRec (20 μM). Rhodopsin phosphorylation by GRK1 in the presence of [γ - 32 P]ATP was monitored at high [Ca 2+ ] free (200 μM Ca 2+ , filled bars) or low [Ca 2+ ] free (0.01 μM, open bars) by phosphorimaging radioautography.

Techniques Used: Functional Assay, Binding Assay, SDS Page, Inhibition, Mutagenesis

35) Product Images from "Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment"

Article Title: Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment

Journal: Communications Biology

doi: 10.1038/s42003-019-0334-5

HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (−) was compared with reducing OPG 22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG 22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG 22–196 and reduced carbamidomethylated OPG 22–196 were detected before and after the incubation with HtrA1 ( m / z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG ( m / z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG 22–90 , OPG 91–196 , m / z 8673 and 12205) derived from OPG 22–196 were detected 5 min after the reaction. c After the incubation of OPG 22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).
Figure Legend Snippet: HtrA1 recognizes the three-dimensional structure and cleaves osteoprotegerin (OPG). a OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT, 10 mM) and iodoacetamide (IAA). Conventional treatment after incubation (−) was compared with reducing OPG 22–196 by pre-treatment with DTT and IAA before incubation (+). OPG fragment sequences were identified using sequence analysis software (Protein Pilot). Amino-acid residues contained in the detected peptides were counted. b OPG 22–196 (2 μg) was incubated with HtrA1 (0.5 μg) at 37 °C for 5 min. The reaction mixture was treated with DTT and IAA, and subjected to MALDI-TOF MS. The measurement of native OPG 22–196 was shown at 0 min. The measurement of reduced carbamidomethylated OPG fragments were shown after the incubation with HtrA1 for 5 min. m/z indicates the mass-to-charge ratio. Intact OPG 22–196 and reduced carbamidomethylated OPG 22–196 were detected before and after the incubation with HtrA1 ( m / z 19778 and 20750, respectively). Before the incubation, a doubly charged ion of OPG ( m / z 9909) was also detected (0 min). Two characteristic OPG fragment peaks (OPG 22–90 , OPG 91–196 , m / z 8673 and 12205) derived from OPG 22–196 were detected 5 min after the reaction. c After the incubation of OPG 22–196 with HtrA1, the reaction mixture was treated with DTT and IAA. The sequences of OPG fragments were identified using Protein Pilot. Detections of the C- and N-terminal residues in OPG fragments were counted. Leucine 90 showed the highest peak at 5 min (arrow).

Techniques Used: Incubation, Sequencing, Software, Mass Spectrometry, Derivative Assay

HtrA1 degrades osteoprotegerin (OPG), whereas MMP9 does not. a Full-length OPG (20 ng) was incubated with HtrA1, mutated HtrA1 (S328A), and MMP9 at 37 °C for 30, 60, and 120 min, and the remaining OPG was detected by western blotting. Amounts of proteases used were also shown. HtrA1 degraded OPG within 30 min, whereas neither mutated HtrA1 (S328A) nor MMP9. b Full-length OPG (2 μg) was incubated with HtrA1, HtrA1 (S328A), and MMP9 (0.5 μg each) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT) and iodoacetamide (IAA), and subjected to mass spectrometry. The sequence of the OPG fragment was identified using sequence analysis software (Protein Pilot, AB SCIEX), and the number of OPG fragments was counted. Degradation of OPG by HtrA1 increased with longer reaction times. c Amino-acid residues contained in peptides detected by mass spectrometry were counted at each reaction time. The positions of the N terminus and C terminus of OPG were 22 and 401, respectively
Figure Legend Snippet: HtrA1 degrades osteoprotegerin (OPG), whereas MMP9 does not. a Full-length OPG (20 ng) was incubated with HtrA1, mutated HtrA1 (S328A), and MMP9 at 37 °C for 30, 60, and 120 min, and the remaining OPG was detected by western blotting. Amounts of proteases used were also shown. HtrA1 degraded OPG within 30 min, whereas neither mutated HtrA1 (S328A) nor MMP9. b Full-length OPG (2 μg) was incubated with HtrA1, HtrA1 (S328A), and MMP9 (0.5 μg each) at 37 °C for the indicated times. The reaction mixture was treated with dithiothreitol (DTT) and iodoacetamide (IAA), and subjected to mass spectrometry. The sequence of the OPG fragment was identified using sequence analysis software (Protein Pilot, AB SCIEX), and the number of OPG fragments was counted. Degradation of OPG by HtrA1 increased with longer reaction times. c Amino-acid residues contained in peptides detected by mass spectrometry were counted at each reaction time. The positions of the N terminus and C terminus of OPG were 22 and 401, respectively

Techniques Used: Incubation, Western Blot, Mass Spectrometry, Sequencing, Software

36) Product Images from "Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten"

Article Title: Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.2003.02130.x

Flavin-hapten-dependent proliferation response of PBMC from two patients with acute myocarditis. PBMC from patients m3 and m4 were stimulated with the FAD-peptide, the FAD-free synthetic peptide with the same amino acid sequence, 6HDNO and the FAD-free 6HDNO.Cys mutant protein. Data are expressed as SI (means and standard deviation).
Figure Legend Snippet: Flavin-hapten-dependent proliferation response of PBMC from two patients with acute myocarditis. PBMC from patients m3 and m4 were stimulated with the FAD-peptide, the FAD-free synthetic peptide with the same amino acid sequence, 6HDNO and the FAD-free 6HDNO.Cys mutant protein. Data are expressed as SI (means and standard deviation).

Techniques Used: Sequencing, Mutagenesis, Standard Deviation

Structure of the naturally occurring FAD-hapten. (a) Covalent attachment of FAD via the 8α-group of the izoaloxazine ring of riboflavin to the N3 nitrogen of a histidine residue of the polypeptide chain. (b) Alignment of the amino acid residues surrounding the FAD-binding His (indicated by a star) of the bacterial flavoenzyme 6HDNO and of the mitochondrial SucDH Fp subunit.
Figure Legend Snippet: Structure of the naturally occurring FAD-hapten. (a) Covalent attachment of FAD via the 8α-group of the izoaloxazine ring of riboflavin to the N3 nitrogen of a histidine residue of the polypeptide chain. (b) Alignment of the amino acid residues surrounding the FAD-binding His (indicated by a star) of the bacterial flavoenzyme 6HDNO and of the mitochondrial SucDH Fp subunit.

Techniques Used: Binding Assay

HPLC purification of the FAD-peptide of 6HDNO. The tryptic digest of 6HDNO and the isolation of the FAD-peptide was performed as described in Material and methods. (a) Tryptic peptide pattern of 6HDNO monitored at 215 nm and 280 nm; (b) the yellow-coloured peptide fraction eluting at 20 min in (a) was rechromatographed and showed a typical flavin absorption at 440 nm.
Figure Legend Snippet: HPLC purification of the FAD-peptide of 6HDNO. The tryptic digest of 6HDNO and the isolation of the FAD-peptide was performed as described in Material and methods. (a) Tryptic peptide pattern of 6HDNO monitored at 215 nm and 280 nm; (b) the yellow-coloured peptide fraction eluting at 20 min in (a) was rechromatographed and showed a typical flavin absorption at 440 nm.

Techniques Used: High Performance Liquid Chromatography, Purification, Isolation

37) Product Images from "Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth ▿"

Article Title: Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01665-08

mTOR S1261 phosphorylation is required for mTORC1's in vivo catalytic activity. (A) In mTORC1, S2481 autophosphorylation increases in response to insulin in a PI3K-dependent manner and upon Rheb overexpression. HEK293 cells were cotransfected with Myc-raptor (0.5 μg) together with Flag-Rheb (2.5 μg) and AU1-mTOR (2.5 μg), as indicated. Cells were serum deprived (24 h), pretreated with wortmannin (W; 100 nM), incubated in the absence or presen ce of insulin (100 nM), and lysed in NP-40-Brij buffer. WCL were immunoprecipitated with Myc antibodies and immunoblotted (IB) as indicated (upper panels). WCL were also immunoblotted (lower panels). (B) In mTORC1, P-S1261 is required for S2481 autophosphorylation. Results are for an experiment similar to that shown in panel A except that additional AU1-mTOR mutant alleles were cotransfected. (C) Phosphorylation of mTOR S1261 does not alter the interaction of mTOR with its TORC1 partners raptor and mLST8/GβL. HEK293 cells were cotransfected with HA-mLST8/GβL (0.5 μg) and HA-raptor (0.5 μg) together with various Myc-mTOR alleles (4 μg), cultured in DMEM-FBS, serum deprived (24 h), incubated in the absence or presence of insulin (100 nM), and lysed in CHAPS buffer. WCL were immunoprecipitated with anti-Myc antibodies and immunoblotted with the indicated antibodies.
Figure Legend Snippet: mTOR S1261 phosphorylation is required for mTORC1's in vivo catalytic activity. (A) In mTORC1, S2481 autophosphorylation increases in response to insulin in a PI3K-dependent manner and upon Rheb overexpression. HEK293 cells were cotransfected with Myc-raptor (0.5 μg) together with Flag-Rheb (2.5 μg) and AU1-mTOR (2.5 μg), as indicated. Cells were serum deprived (24 h), pretreated with wortmannin (W; 100 nM), incubated in the absence or presen ce of insulin (100 nM), and lysed in NP-40-Brij buffer. WCL were immunoprecipitated with Myc antibodies and immunoblotted (IB) as indicated (upper panels). WCL were also immunoblotted (lower panels). (B) In mTORC1, P-S1261 is required for S2481 autophosphorylation. Results are for an experiment similar to that shown in panel A except that additional AU1-mTOR mutant alleles were cotransfected. (C) Phosphorylation of mTOR S1261 does not alter the interaction of mTOR with its TORC1 partners raptor and mLST8/GβL. HEK293 cells were cotransfected with HA-mLST8/GβL (0.5 μg) and HA-raptor (0.5 μg) together with various Myc-mTOR alleles (4 μg), cultured in DMEM-FBS, serum deprived (24 h), incubated in the absence or presence of insulin (100 nM), and lysed in CHAPS buffer. WCL were immunoprecipitated with anti-Myc antibodies and immunoblotted with the indicated antibodies.

Techniques Used: In Vivo, Activity Assay, Over Expression, Incubation, Immunoprecipitation, Mutagenesis, Cell Culture

Phosphorylation of mTOR S1261 promotes mTORC1-mediated 4EBP1 phosphorylation. HEK293 cells were transfected with various AU1-mTOR alleles (4 μg) together with 3HA-4EBP1 (1 μg), deprived of serum (24 h), pretreated without or with rapamycin (20 ng/ml) for 30 min, incubated in the absence or presence of insulin (100 nM) for 30 min, and lysed in NP-40-Brij buffer. WCL were resolved on SDS-PAGE and immunoblotted with the indicated antibodies. SE, short exposure; LE, long exposure. The positions of 3HA-4EBP1 (*) versus endogenous 4EBP1 (**) are indicated.
Figure Legend Snippet: Phosphorylation of mTOR S1261 promotes mTORC1-mediated 4EBP1 phosphorylation. HEK293 cells were transfected with various AU1-mTOR alleles (4 μg) together with 3HA-4EBP1 (1 μg), deprived of serum (24 h), pretreated without or with rapamycin (20 ng/ml) for 30 min, incubated in the absence or presence of insulin (100 nM) for 30 min, and lysed in NP-40-Brij buffer. WCL were resolved on SDS-PAGE and immunoblotted with the indicated antibodies. SE, short exposure; LE, long exposure. The positions of 3HA-4EBP1 (*) versus endogenous 4EBP1 (**) are indicated.

Techniques Used: Transfection, Incubation, SDS Page

Identification of S1261 as a novel in vivo mTOR phosphorylation site in intact cells. (A) mTOR undergoes extensive phosphorylation in vivo. HEK293 cells cultured in DMEM-FBS (steady state) were lysed in CHAPS buffer, and mTOR was immunoprecipitated. Immunoprecipitates were either resuspended immediately in sample buffer (IP) or washed in phosphatase buffer and incubated in vitro with various units of λ-phosphatase, resolved on 6% SDS-PAGE, and immunoblotted as indicated (IB). (B) Low-energy collision-induced dissociation spectrum of the doubly charged mTOR pS1261 phosphopeptide. HEK293 cells were transfected with AU1-mTOR, cultured in DMEM-FBS, lysed in CHAPS buffer, and immunoprecipitated with AU1 antibodies. A Coomassie-stained band of AU1-mTOR was digested with trypsin after SDS-PAGE. Liquid chromatography-MS/MS and data analysis were conducted as described in Materials and Methods. Note that the y 6 ). The tryptic peptide in which P-S1261 was identified is shown. (D) Alignment of mTOR S1261 from various organisms using the algorithm Clustal W. The Caenorhabditis elegans sequence was omitted due to poor alignment resulting from large regions of nonhomology. (E) P-S1261 antibodies are site specific. HEK293 cells were transfected with vector control (V) or with WT or S1261A AU1-mTOR alleles, as indicated, and lysed in CHAPS buffer. WCL were immunoprecipitated with AU1 antibodies and immunoblotted with the indicated antibodies. (F) P-S1261 antibodies are phospho-specific. mTOR immunoprecipitates from HEK293 cells (lysed in CHAPS buffer) were either resuspended immediately in sample buffer (IP) or washed in phosphatase buffer and incubated in the absence or presence of λ-phosphatase (250 units). The immunoprecipitates were immunoblotted as indicated. (G) S1261 is not an autophosphorylation site. HEK293 cells were transfected with vector control, WT (1.0 μg), or KD (1.0 μg) Myc-mTOR plasmids and incubated in the absence or presence of rapamycin (20 ng/ml) for 2 h. Cells were lysed in CHAPS buffer, and WCLs were immunoprecipitated with Myc antibodies and immunoblotted as indicated.
Figure Legend Snippet: Identification of S1261 as a novel in vivo mTOR phosphorylation site in intact cells. (A) mTOR undergoes extensive phosphorylation in vivo. HEK293 cells cultured in DMEM-FBS (steady state) were lysed in CHAPS buffer, and mTOR was immunoprecipitated. Immunoprecipitates were either resuspended immediately in sample buffer (IP) or washed in phosphatase buffer and incubated in vitro with various units of λ-phosphatase, resolved on 6% SDS-PAGE, and immunoblotted as indicated (IB). (B) Low-energy collision-induced dissociation spectrum of the doubly charged mTOR pS1261 phosphopeptide. HEK293 cells were transfected with AU1-mTOR, cultured in DMEM-FBS, lysed in CHAPS buffer, and immunoprecipitated with AU1 antibodies. A Coomassie-stained band of AU1-mTOR was digested with trypsin after SDS-PAGE. Liquid chromatography-MS/MS and data analysis were conducted as described in Materials and Methods. Note that the y 6 ). The tryptic peptide in which P-S1261 was identified is shown. (D) Alignment of mTOR S1261 from various organisms using the algorithm Clustal W. The Caenorhabditis elegans sequence was omitted due to poor alignment resulting from large regions of nonhomology. (E) P-S1261 antibodies are site specific. HEK293 cells were transfected with vector control (V) or with WT or S1261A AU1-mTOR alleles, as indicated, and lysed in CHAPS buffer. WCL were immunoprecipitated with AU1 antibodies and immunoblotted with the indicated antibodies. (F) P-S1261 antibodies are phospho-specific. mTOR immunoprecipitates from HEK293 cells (lysed in CHAPS buffer) were either resuspended immediately in sample buffer (IP) or washed in phosphatase buffer and incubated in the absence or presence of λ-phosphatase (250 units). The immunoprecipitates were immunoblotted as indicated. (G) S1261 is not an autophosphorylation site. HEK293 cells were transfected with vector control, WT (1.0 μg), or KD (1.0 μg) Myc-mTOR plasmids and incubated in the absence or presence of rapamycin (20 ng/ml) for 2 h. Cells were lysed in CHAPS buffer, and WCLs were immunoprecipitated with Myc antibodies and immunoblotted as indicated.

Techniques Used: In Vivo, Cell Culture, Immunoprecipitation, Incubation, In Vitro, SDS Page, Transfection, Staining, Liquid Chromatography, Mass Spectrometry, Sequencing, Plasmid Preparation

38) Product Images from "Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals"

Article Title: Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1604463113

Details of the Mm α-GluII α-subunit active site at key stages during its catalytic cycle (with corresponding chemical schemes shown in the upper right corners). The −1 and +1 subsites are marked in B . ( A ) Apo enzyme; the α-subunit
Figure Legend Snippet: Details of the Mm α-GluII α-subunit active site at key stages during its catalytic cycle (with corresponding chemical schemes shown in the upper right corners). The −1 and +1 subsites are marked in B . ( A ) Apo enzyme; the α-subunit

Techniques Used:

SAXS and crystal structures of Mm α-GluII. ( A ) A 1-Dimensional representation of the α-GluII subunits. The trypsinolysis sites are symbolized by scissors, and portions of the α-subunit removed by trypsin are represented by magenta
Figure Legend Snippet: SAXS and crystal structures of Mm α-GluII. ( A ) A 1-Dimensional representation of the α-GluII subunits. The trypsinolysis sites are symbolized by scissors, and portions of the α-subunit removed by trypsin are represented by magenta

Techniques Used:

α-Subunit N terminus extension and exclusion loop insertion are previously unidentified α-GluII-specific determinants of enzyme activity. ( A ) Mm α-GluII Tryps α-subunit (green) with its bound disaccharide substrate analog
Figure Legend Snippet: α-Subunit N terminus extension and exclusion loop insertion are previously unidentified α-GluII-specific determinants of enzyme activity. ( A ) Mm α-GluII Tryps α-subunit (green) with its bound disaccharide substrate analog

Techniques Used: Activity Assay

Crystal structures of Mm α-GluII Tryps in complex with iminosugar inhibitors. Structures of complexes of Mm α-GluII Tryps with castanospermine ( A ), DNJ ( B ), N B-DNJ ( C ), and M ON -DNJ ( D ). Upon alkylation of the endocyclic nitrogen, the W525
Figure Legend Snippet: Crystal structures of Mm α-GluII Tryps in complex with iminosugar inhibitors. Structures of complexes of Mm α-GluII Tryps with castanospermine ( A ), DNJ ( B ), N B-DNJ ( C ), and M ON -DNJ ( D ). Upon alkylation of the endocyclic nitrogen, the W525

Techniques Used:

39) Product Images from "Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay"

Article Title: Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay

Journal: Reproductive biology and endocrinology : RB & E

doi: 10.1186/1477-7827-3-49

GnSAF immunopurified from G/LCM . Immunopurification using phage-derived antibody immobilised on protein-L agarose (a,b) or rat polyclonal antibody immobilised on anti-rat IgG-coated magnetic beads (c,d) was pooled after 15 consecutive loading and elution cycles with 2 M NaI. Proteins detected by Coomassie blue staining of (a,c) 2-D (immunopurified protein only, numbers match those in Table II) and (b, d) 1-D (Unbound = flow-through, Bound = immunopurified) gels were investigated by mass spectroscopic peptide mass mapping. Western blot of the 3-c4b-immunopurified G/LCM (e,f) with either 3-c4b and secondary antibody (e) or secondary antibody only (f) show the elution of a specifically recognised 60–70 kDa protein band (arrow) by 2 M NaI, with no further protein eluted with glycine washes.
Figure Legend Snippet: GnSAF immunopurified from G/LCM . Immunopurification using phage-derived antibody immobilised on protein-L agarose (a,b) or rat polyclonal antibody immobilised on anti-rat IgG-coated magnetic beads (c,d) was pooled after 15 consecutive loading and elution cycles with 2 M NaI. Proteins detected by Coomassie blue staining of (a,c) 2-D (immunopurified protein only, numbers match those in Table II) and (b, d) 1-D (Unbound = flow-through, Bound = immunopurified) gels were investigated by mass spectroscopic peptide mass mapping. Western blot of the 3-c4b-immunopurified G/LCM (e,f) with either 3-c4b and secondary antibody (e) or secondary antibody only (f) show the elution of a specifically recognised 60–70 kDa protein band (arrow) by 2 M NaI, with no further protein eluted with glycine washes.

Techniques Used: Laser Capture Microdissection, Immu-Puri, Derivative Assay, Magnetic Beads, Staining, Flow Cytometry, Western Blot

40) Product Images from "Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50Cdc37 complex and direct interactions with amino acids residues of Hsp90"

Article Title: Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50Cdc37 complex and direct interactions with amino acids residues of Hsp90

Journal: The Journal of nutritional biochemistry

doi: 10.1016/j.jnutbio.2011.11.004

Influence of sulforaphane on ATP binding of Hsp90 and Hsp90-cochaperone association in Mia Paca-2 cells. (a) Sulforaphane (5, 15, 30 μM) did not affect ATP binding, while 17-AAG (5 μM) decreased ATP binding to Hsp90. (b) Sulforaphane reduced
Figure Legend Snippet: Influence of sulforaphane on ATP binding of Hsp90 and Hsp90-cochaperone association in Mia Paca-2 cells. (a) Sulforaphane (5, 15, 30 μM) did not affect ATP binding, while 17-AAG (5 μM) decreased ATP binding to Hsp90. (b) Sulforaphane reduced

Techniques Used: Binding Assay

Sulforaphane exhibits anticancer activity in vitro and in vivo . (a) Sulforaphane inhibited proliferation of Mia Paca-2, Panc-1, AsPc-1, and BxPc-3 cells. (b) Sulforaphane induced caspase-3 activity in Mia Paca-2 cells. Data are presented as means ±
Figure Legend Snippet: Sulforaphane exhibits anticancer activity in vitro and in vivo . (a) Sulforaphane inhibited proliferation of Mia Paca-2, Panc-1, AsPc-1, and BxPc-3 cells. (b) Sulforaphane induced caspase-3 activity in Mia Paca-2 cells. Data are presented as means ±

Techniques Used: Activity Assay, In Vitro, In Vivo

Sulforaphane induces proteasomal degradation of Hsp90 client proteins. (a) Mia Paca-2 and Panc-1 cells were treated with 15 μM sulforaphane for different time periods. Sulforaphane induced a time-dependent down-regulation of Akt, Cdk4, and p53
Figure Legend Snippet: Sulforaphane induces proteasomal degradation of Hsp90 client proteins. (a) Mia Paca-2 and Panc-1 cells were treated with 15 μM sulforaphane for different time periods. Sulforaphane induced a time-dependent down-regulation of Akt, Cdk4, and p53

Techniques Used:

Sulforaphane binding to Hsp90 mapped by NMR. (a) Rainbow representation of 1 H- 13 C-Ile methyl TROSY cross peaks of full length Hsp90 bound to sulforaphane (1.5 mM) (blue to red gradient indicates increasing intensities; peak centers, black dots). (b) Overlayed
Figure Legend Snippet: Sulforaphane binding to Hsp90 mapped by NMR. (a) Rainbow representation of 1 H- 13 C-Ile methyl TROSY cross peaks of full length Hsp90 bound to sulforaphane (1.5 mM) (blue to red gradient indicates increasing intensities; peak centers, black dots). (b) Overlayed

Techniques Used: Binding Assay, Nuclear Magnetic Resonance

Proteolytic fingerprinting assay and LC-MS analysis of Hsp90 interaction with sulforaphane. (a) After incubation with DMSO or sulforaphane, protein sample (Hsp90βN) was digested with the indicated concentrations of trypsin. Hsp90 antibody (N-17),
Figure Legend Snippet: Proteolytic fingerprinting assay and LC-MS analysis of Hsp90 interaction with sulforaphane. (a) After incubation with DMSO or sulforaphane, protein sample (Hsp90βN) was digested with the indicated concentrations of trypsin. Hsp90 antibody (N-17),

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation

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Incubation:

Article Title: H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase
Article Snippet: .. Thermolysin (Promega) was added at a protein mass ratio of 1:20 thermolysin:RAG and incubation was continued at 37 °C for 15 min. .. Samples were removed at 5, 10, and 15 min and reactions were stopped by heating to 100 °C in SDS/PAGE loading buffer containing 50 mM EDTA.

Article Title: Proteome Analysis of Potato Starch Reveals the Presence of New Starch Metabolic Proteins as Well as Multiple Protease Inhibitors
Article Snippet: .. 60 μg of thermolysin (Promega, France) were added to 300 mg of starch in 50 mM Tris, 0.5 mM CaCl2 , pH 8.0 prior to incubation overnight at 37°C. .. Starch granules were then washed five times with 2 mL of ultrapure water and protein extraction was carried out with the addition of 5 mL of extraction buffer (0.2 M Tris, 2% SDS, 20% glycerol, 50 mM DTT, pH 6.8) and subsequent incubation at 100°C for 20 min with regular vortexing.

Binding Assay:

Article Title: Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein
Article Snippet: .. To assess binding of mAbs to Jurkat-EBOV GPCL , Jurkat-EBOV GP cells were counted and treated with 0.5 mg/mL of thermolysin (Promega) in PBS for 20 min at 37°C. .. Cell staining and flow cytometric analysis was performed as described above.

Article Title: Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization
Article Snippet: .. To assess binding of mAbs to cleaved GP, Jurkat-EBOV, -BDBV, or -SUDV GP cells were treated with 0.5 mg/mL of thermolysin (Promega) in PBS for 20 min at 37°C. .. Cells staining and flow cytometric analysis was performed as described above.

Purification:

Article Title: Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap
Article Snippet: .. VSV/BDBV-GP purified as described above was resuspended in thermolysin digestion buffer (50 mM Tris, pH 8.0, 0.5 mM CaCl2 ) and divided into two aliquots; one aliquot was treated with 0.5 mg/ml of thermolysin (Promega) and another one with an equal volume of thermolysin digestion buffer (mock-treated virus) for 40 min at 37ºC. .. The reactions were stopped by addition of EDTA up to the final concentration 10 mM.

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