Journal: Cell Death & Disease

Article Title: Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

doi: 10.1038/cddis.2010.67

Figure Lengend Snippet: Effects of ESOM on autophagic flux. ( a ) Me30966 cells treated with ESOM (160 μ M) for 8 h are characterized by massive intracellular vacuolization. ( b ) Treatment with ESOM (for 8 h) induces accumulation of LC3-positive autophagic vacuoles in Me30966 and WM793 cells. ( c ) ESOM induces accumulation of the LC3-II form in autophagy-competent melanoma cells, as shown by the further accumulation of LC3-II in cells pretreated with inhibitors of lysosomal proteases. ( d ) ESOM-induced alteration of the autophagic flux is confirmed by accumulation of p62. ( e and f ) Analysis of the mRFP-GFP-LC3-plasmid shows the accumulation of autophagosomes in ESOM-treated Me30966 cells. Cells cultured in EBSS were used as positive control

Article Snippet: Anti-LC3 antibody against a synthetic LC3 peptide was produced in rabbits by NeoMPS (Strasbourg, France).

Techniques: Plasmid Preparation, Cell Culture, Positive Control

Journal: Cell Death & Disease

Article Title: Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

doi: 10.1038/cddis.2010.67

Figure Lengend Snippet: Role of ROS and mTOR in ESOM effects on the autophagic flux. ( a and b ) NAC pretreatment prevents the accumulation of autophagosomes induced by ESOM (160 μ M) in Me30966 cells, as shown by confocal fluorescence analysis of endogenous LC3 and quantification of cells with LC3+ dots. ( c ) Western blot analysis of LC3-I and LC3-II in the presence or absence of NAC in ESOM-treated cells. ( d and e ) ESOM treatment inhibits the mTOR pathway as shown by the time-dependent reduced phosphorylation of 4-EBP1 and p70S6K proteins. Such inhibition is prevented by the ROS scavenger NAC

Article Snippet: Anti-LC3 antibody against a synthetic LC3 peptide was produced in rabbits by NeoMPS (Strasbourg, France).

Techniques: Fluorescence, Western Blot, Phospho-proteomics, Inhibition

Journal: Cell Death & Disease

Article Title: Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

doi: 10.1038/cddis.2010.67

Figure Lengend Snippet: Autophagy is a cytoprotective mechanism in ESOM-treated melanoma cells. ( a ) Me30966 cells pretreated with Baf-A1 (40 nM) show increased sensitivity to ESOM-induced apoptosis in association with a further increased LC3-II accumulation. ( b ) Knockdown of Atg5 and beclin-1 induces a cytosolic localization of LC3 as compared with autophagosomal localization in control Me30966 cells. ( c and d ) Knockdown of Atg5 and beclin-1 increases the cytotoxicity of ESOM in Me30966 cells and WM793 cells, respectively

Article Snippet: Anti-LC3 antibody against a synthetic LC3 peptide was produced in rabbits by NeoMPS (Strasbourg, France).

Techniques: Knockdown, Control

Journal: BMC Immunology

Article Title: Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

doi: 10.1186/1471-2172-10-13

Figure Lengend Snippet: Truncated CHIPS variants inhibit C5a induced cell activation . Fluo-3AM labeled U937/C5aR transfectants were preincubated with full-length CHIPS 1–121 , CHIPS with N-terminal truncation (CHIPS ΔN), CHIPS with C-terminal truncation (CHIPS ΔC) and CHIPS with truncations in both the N- and C-terminal ends (CHIPS ΔN/C) and stimulated with 1 nM C5a. Results are expressed as percentage inhibition of buffer treated cells and are from a representative experiment.

Article Snippet: A 38-mer peptide comprising the N-terminal part of CHIPS (first 37 amino acids plus an additional cysteine (Pepscan, Lelystad, The Netherlands) served as control.

Techniques: Activation Assay, Labeling, Inhibition

Journal: BMC Immunology

Article Title: Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

doi: 10.1186/1471-2172-10-13

Figure Lengend Snippet: Affinity-purified anti-CHIPS IgG recognize non-linear epitopes . Affinity-purified anti-CHIPS 1–121 IgG was tested for the ability to bind CHIPS derived 25-mer peptides or full-length CHIPS in ELISA (A). Full-length CHIPS 1–121 or CHIPS with N-terminal truncation (CHIPS ΔN), or CHIPS with truncations in both the N- and C-terminal ends (CHIPS ΔN/C) were compared for reactivity with different human affinity-purified anti-full-length CHIPS 1–121 IgG (B), anti-CHIPS ΔN/C IgG (C), and the CHIPS specific mouse monoclonal antibody 2G8 as control (D).

Article Snippet: A 38-mer peptide comprising the N-terminal part of CHIPS (first 37 amino acids plus an additional cysteine (Pepscan, Lelystad, The Netherlands) served as control.

Techniques: Affinity Purification, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: BMC Immunology

Article Title: Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

doi: 10.1186/1471-2172-10-13

Figure Lengend Snippet: Inhibition of C5aR activity by CHIPS variants with C-terminal truncation . Fluo-3AM labeled U937/C5aR transfectants were preincubated with CHIPS variants and stimulated with C5a. Results are expressed as percentage inhibition of buffer treated cells.

Article Snippet: A 38-mer peptide comprising the N-terminal part of CHIPS (first 37 amino acids plus an additional cysteine (Pepscan, Lelystad, The Netherlands) served as control.

Techniques: Inhibition, Activity Assay, Labeling

Journal: BMC Immunology

Article Title: Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

doi: 10.1186/1471-2172-10-13

Figure Lengend Snippet: CHIPS with C-terminal truncation and selected corresponding mutants have the same overall structure . CD spectra of C-terminally truncated CHIPS and variants with reduced C5aR blocking activity, carrying one of the amino acid substitutions K95S, Y97K, K100A or S107N, were recorded. All CHIPS variants have a significant absorption minimum at 205 nm and are suggested to have the same overall structure as the corresponding wt protein.

Article Snippet: A 38-mer peptide comprising the N-terminal part of CHIPS (first 37 amino acids plus an additional cysteine (Pepscan, Lelystad, The Netherlands) served as control.

Techniques: Circular Dichroism, Blocking Assay, Activity Assay

Journal: Nature Communications

Article Title: Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank–Starling response

doi: 10.1038/ncomms13187

Figure Lengend Snippet: ( a ) Cardiac substrates of PKGIα were identified by a chemical genetic phosphoproteomic method and the amount of substrate phosphorylation was quantitated when PKGIα was in its basal state (control), activated by disulfide bond, or activated by cGMP. The scatter plot shows the PKGIα activation-dependent log 2 fold changes in phosphopeptide abundance relative to control. Pink circles represent phosphopeptides whose abundances were significantly increased compared with control ( P <0.05; n =4) when PKGIα activity was stimulated by cGMP and the blue circle represents a phosphopeptide whose abundance was significantly increased relative to control ( P <0.05; n =4) when PKGIα was activated by disulfide; this phosphopeptide, RAS(p)TIEMPQQAR, is from the cardiac SR protein phospholamban (PLN) and is phosphorylated at residue Ser16. Decreases in phosphorylation were not statistically significant for any substrate. P values were determined by post hoc Dunnett's test following one-way ANOVA. ( b ) Oxidized PKGIα-mediated phosphorylation of PLN was confirmed by a study of the basal level of Ser16 phosphorylation in isolated hearts from C42S PKGIα KI mice which cannot form the activating disulfide bond. PLN Ser16 phosphorylation was significantly decreased in the KI compared with WT (* P <0.05; n =6) while phosphorylation of Thr17 was unchanged. We also observed a significant three-fold increase in the pentamer/monomer ratio of total PLN in the KI hearts, indicating that the oligomeric state of PLN is also affected by the oxidation state of PKGIα. ( c ) Immunoblots from WT or KI myocardium for several other key proteins and their phosphosites involved in cardiac EC coupling and Ca 2+ handling. No significant changes in phosphorylation or total protein levels were detected between genotypes. Histograms show the mean±s.e.m. and P values were determined by t -test.

Article Snippet: A synthetic N-terminally acetylated peptide corresponding to the N-terminal cytoplasmic domain of human phospholamban, PLN (amino acid 1-23) was purchased from PeptideSynthetics (purity >95%; Peptide Protein Research Ltd, UK).

Techniques: Phospho-proteomics, Control, Activation Assay, Activity Assay, Residue, Isolation, Western Blot

Journal: Nature Communications

Article Title: Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank–Starling response

doi: 10.1038/ncomms13187

Figure Lengend Snippet: ( a ) Curves showing the variation in SP, rate of contraction (+d p /d t ), and rate of relaxation (−d p /d t ) as a function of EDP for Langendorff-perfused WT and KI hearts. Cardiac performance increased with EDP, that is, stretch, according to the Frank–Starling law. However, the responses were significantly reduced in the KI hearts compared with WT ( P <0.05; n =8). ( b ) Immunoblotting showed that oxidation of PKGIα to the disulfide dimer increased with increasing stretch (from 0 mm Hg to 5 mm Hg EDP) in the WT hearts but not in the KI (* P <0.05; n =5). ( c ) Phosphorylation of PLN Ser16 was also significantly increased in the WT but not KI hearts with increased diastolic stretch (* P <0.05; n =5). ( d ) Subcellular fractionation of WT and KI hearts perfused at different EDPs followed by immunoblotting revealed a significant increase in the amount of PKGIα in the particulate fraction from stretched WT myocardium compared with the particulate fraction from unstretched WT myocardium (* P <0.05; n =5). No stretch-dependent changes in PKGIα abundance were observed in fractions from the KI tissue. Error bars show s.e.m. and P values were determined by t -test. ( e ) ITC analysis of the interaction between the cytoplasmic domain of PLN (residues 1–23) with disulfide-activated PKGIα and the C42S mutant. A sigmoidal binding isotherm can be fitted to the titration data for oxidized PKGIα which is consistent with one PKGIα disulfide dimer binding to one PLN peptide with a K d of ∼7 μM. Although the ITC data for C42S PKGIα also suggests a direct interaction with the cytoplasmic domain of PLN, this is markedly weaker than for oxidized PKGIα as the integrated data cannot be fitted to a sigmoid-shaped binding curve under the same experimental conditions.

Article Snippet: A synthetic N-terminally acetylated peptide corresponding to the N-terminal cytoplasmic domain of human phospholamban, PLN (amino acid 1-23) was purchased from PeptideSynthetics (purity >95%; Peptide Protein Research Ltd, UK).

Techniques: Western Blot, Phospho-proteomics, Fractionation, Mutagenesis, Binding Assay, Titration