Journal: American Journal of Physiology - Renal Physiology

Article Title: Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome

doi: 10.1152/ajprenal.00207.2017

Figure Lengend Snippet: ADR-induced injury induced and activated small heat shock proteins (HSPs), primarily in an MK2-dependent manner. Total protein was extracted from renal cortexes isolated from control and ADR-injured mice on day 21 from each genotype and phosphorylated and total forms of HSPB1, HSPB8, glucose-regulated protein 78 (GRP78), and GAPDH were detected using their respective antibodies. A: representative Western blots of total and phosphorylated forms of selected proteins. B: densitometry analyses of protein induction/phosphorylation in the renal cortexes of at least 3 mice from each genotype. Statistical differences: *P < 0.05, control vs. ADR; #P < 0.05, WT-ADR treated vs. KO-ADR treated.

Article Snippet: The other primary antibodies used were anti-HSPB1 (StressMarq, Victoria, BC), anti-HSPB8 mouse monoclonal (Abcam, Cambridge, MA), anti-GRP78 rabbit monoclonal (StressMarq), and anti-GAPDH mouse monoclonal (Millipore, Billerica, MA).

Techniques: Isolation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome

doi: 10.1152/ajprenal.00207.2017

Figure Lengend Snippet: Schematic summarizing and depicting the roles of MK2 and MK3 in regulating stress response and proteinuria during nephrotic syndrome nephrotic syndrome induction in experimental rodent models resulted in massive proteinuria. Genetic deletion of MK2 and MK3 did not impart any clear proteinuria reducing effects in ADR-induced nephrotic syndrome (NS) in mice. Similarly, specific pharmacological MK2 inhibition also did not result in significant proteinuria reducing effects in PAN-induced NS. We previously reported similar nonbeneficial effects of MK2 and MK3 deletion in acute proliferative glomerulonephritis model in mice (20). Interestingly, we found that ADR injury resulted in massive renal cortical induction of the sHSPs HSPB1 and HSPB8, while HSPB1 was also significantly phosphorylated and activated only in the genotypes WT for MK2 (WT and MK3−/−) and only very mildly activated in the genotypes deleted for MK2 and WT for MK3 (MK3−/− and MK2−/−MK3−/−). These results suggest that while MK2 and MK3 inhibition may not induce clear reductions in proteinuria during NS, MK2, and not MK3, plays an important role in mediating the renal stress response in a chronic model of NS. Together, these and our previous preclinical studies suggest that our currently available approaches are not yet able to safely and effectively reduce proteinuria in experimental NS and that other p38 MAPK downstream targets (i.e., other cytosolic substrates, chromatin remodelers, and transcription factors) (10, 18) should also be considered to improve the future treatment of glomerular disease.

Article Snippet: The other primary antibodies used were anti-HSPB1 (StressMarq, Victoria, BC), anti-HSPB8 mouse monoclonal (Abcam, Cambridge, MA), anti-GRP78 rabbit monoclonal (StressMarq), and anti-GAPDH mouse monoclonal (Millipore, Billerica, MA).

Techniques: Inhibition

Journal: eLife

Article Title: Hsf1 and the molecular chaperone Hsp90 support a ‘rewiring stress response’ leading to an adaptive cell size increase in chronic stress

doi: 10.7554/eLife.88658

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Hsp25/27 (Mouse monoclonal) , StressMarq , SMC-114 , 1:1000.

Techniques: Generated, Recombinant, Plasmid Preparation, Over Expression, Mutagenesis, Control, Sequencing, shRNA, Flow Cytometry

Journal: PLoS ONE

Article Title: Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis

doi: 10.1371/journal.pone.0054239

Figure Lengend Snippet: Activation of the p38 MAPK by various stress stimuli or growth factors results in activation of MK2 and MK3. The signal transduction by these protein kinases towards the major substrates tristetraprolin (TTP) and HSPB1 is typically additive, although in most cells MK2 is the prevailing signal transducer, with little contribution of MK3. MK5 is activated by PKA, probably independently of p38 MAPK. The role of putative MK5 becomes obvious in the absence of MK2 and MK3, as shown in the present study. MK2, MK3, and MK5 phosphorylate the same two sites in mouse HSPB1 (Ser15, Ser86). In addition to MK2 and MK3, MNK1 and MSK1/2 are further MKs that are downstream of p38 MAPK. In macrophages, MK2 and MK3 were found to control expression of the immune response mediators IFNβ, IL-10, and NFκB through regulation of the activity of IRF3 and IκBβ. In these cells, MK2 was demonstrated to prevent MK3 from exerting negative regulatory effects on IRF3- and NFκB-dependent signaling. Dashed arrows indicate indirect signal transduction, while open arrows indicate complex effects on biological responses.

Article Snippet: The following primary antibodies (Ab) were used: anti-MK2 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling, Danvers, MA), anti-MK3 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-β-actin rabbit monoclonal Ab (dilution: 1∶5000; Cell Signaling), anti-eIF2α mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-phospho-eIF2α rabbit monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-CHOP mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-HSPB1 rabbit polyclonal Ab (dilution: 1∶5000; Assay Designs), anti-phospho-HSPB1-Ser86 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling), anti-GRP78 rabbit polyclonal Ab (dilution: 1∶1000; StressMarq, Victoria, BC), anti-Hsp70 mouse monoclonal Ab (dilution: 1∶1000; StressMarq), anti-HSPB8 mouse monoclonal Ab (dilution: 1∶1000; Abcam, Cambridge, MA), anti-HSPB6 rabbit polyclonal Ab (dilution: 1∶1000; Abcam), anti-MK5 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-MK5 rabbit polyclonal Ab (dilution 1∶500; Pierce Biotechnology, Rockford, IL), anti-MNK1 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-Mnk1 polyclonal Ab (dilution 1∶1000; Cell Signaling), anti-MSK1 rabbit polyclonal Ab (dilution 1∶500; LifeSpan BioSciences, Seattle, WA), anti-phospho-MSK1 rabbit polyclonal Ab (dilution 1∶500; R&D Systems, Minneapolis, MN), and anti-GAPDH mouse monoclonal Ab (dilution 1∶5000; Millipore, Billerica, MA).

Techniques: Activation Assay, Transduction, Control, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis

doi: 10.1371/journal.pone.0054239

Figure Lengend Snippet: Extracts of renal cortices were processed for IEF-PAGE (A, panel a) or SDS-PAGE (A, panels b, c; B, C, D) from untreated mice (day 0; baseline control) and AMC serum-treated mice (day 8 of treatment). (A) Phosphorylation, baseline expression and induction of HSPB1. Panel a shows the distribution of the various HSPB1 isoforms (0p, unphosphorylated; 1p, singly phosphorylated; 2p, doubly phosphorylated) within each sample. Sample loading aimed to obtain comparable overall signals, in spite of considerable differences in the total HSPB1 content among the samples (cf. panel c). Panel b shows the amounts of Ser86-phosphorylated HSPB1 (p-Ser86). Equal amounts of total protein (15 µg) were loaded onto each lane. Panel c shows baseline expression and induction of HSPB1 in response to the AMC serum. (B) Baseline expression and response to the AMC serum of the heat shock proteins, HSPB6, HSPB8, and HSP70 (panels a–c, respectively). (C) Expression or phosphorylation of indicators of the unfolded protein response, CHOP (panel a), GRP78 (panel b), and eIF2α (panels c, d), before and after AMC serum treatment. Panels c and d show phosphorylated (p-eIF2α) and total eIF2α, respectively. (D) Expression of β-actin served as a loading control. Overall, this figure demonstrates partial involvement of MK2 and MK3 in baseline expression and/or phosphorylation of a number of sHSPs and indicators of the unfolded protein response, as well as in their pathophysiological response following AMC serum treatment.

Article Snippet: The following primary antibodies (Ab) were used: anti-MK2 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling, Danvers, MA), anti-MK3 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-β-actin rabbit monoclonal Ab (dilution: 1∶5000; Cell Signaling), anti-eIF2α mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-phospho-eIF2α rabbit monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-CHOP mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-HSPB1 rabbit polyclonal Ab (dilution: 1∶5000; Assay Designs), anti-phospho-HSPB1-Ser86 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling), anti-GRP78 rabbit polyclonal Ab (dilution: 1∶1000; StressMarq, Victoria, BC), anti-Hsp70 mouse monoclonal Ab (dilution: 1∶1000; StressMarq), anti-HSPB8 mouse monoclonal Ab (dilution: 1∶1000; Abcam, Cambridge, MA), anti-HSPB6 rabbit polyclonal Ab (dilution: 1∶1000; Abcam), anti-MK5 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-MK5 rabbit polyclonal Ab (dilution 1∶500; Pierce Biotechnology, Rockford, IL), anti-MNK1 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-Mnk1 polyclonal Ab (dilution 1∶1000; Cell Signaling), anti-MSK1 rabbit polyclonal Ab (dilution 1∶500; LifeSpan BioSciences, Seattle, WA), anti-phospho-MSK1 rabbit polyclonal Ab (dilution 1∶500; R&D Systems, Minneapolis, MN), and anti-GAPDH mouse monoclonal Ab (dilution 1∶5000; Millipore, Billerica, MA).

Techniques: SDS Page, Control, Phospho-proteomics, Expressing

Journal: PLoS ONE

Article Title: Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis

doi: 10.1371/journal.pone.0054239

Figure Lengend Snippet: Paraffin-embedded renal cortices of untreated and AMC serum-treated wild-type and MK2/MK3 double knock-out mice (day 16 following AMC serum treatment) were sectioned and processed for immunofluorescence microscopy. Total HSPB1 was visualized using an anti-HSPB1 antibody. In untreated control mice of either genotype, labeling of the glomeruli (including Bowman's space) was moderately elevated as compared to the surrounding tubules (upper row, left panels) or to the more distant tubules (upper row, right panels). AMC serum treatment caused a strong increase in HSPB1 labeling in the tubules, both adjacent to the glomeruli (lower row, left panels) and more distant from the glomeruli (lower row, right panels), thus indicating a stress response in the tubular compartment.

Article Snippet: The following primary antibodies (Ab) were used: anti-MK2 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling, Danvers, MA), anti-MK3 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-β-actin rabbit monoclonal Ab (dilution: 1∶5000; Cell Signaling), anti-eIF2α mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-phospho-eIF2α rabbit monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-CHOP mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-HSPB1 rabbit polyclonal Ab (dilution: 1∶5000; Assay Designs), anti-phospho-HSPB1-Ser86 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling), anti-GRP78 rabbit polyclonal Ab (dilution: 1∶1000; StressMarq, Victoria, BC), anti-Hsp70 mouse monoclonal Ab (dilution: 1∶1000; StressMarq), anti-HSPB8 mouse monoclonal Ab (dilution: 1∶1000; Abcam, Cambridge, MA), anti-HSPB6 rabbit polyclonal Ab (dilution: 1∶1000; Abcam), anti-MK5 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-MK5 rabbit polyclonal Ab (dilution 1∶500; Pierce Biotechnology, Rockford, IL), anti-MNK1 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-Mnk1 polyclonal Ab (dilution 1∶1000; Cell Signaling), anti-MSK1 rabbit polyclonal Ab (dilution 1∶500; LifeSpan BioSciences, Seattle, WA), anti-phospho-MSK1 rabbit polyclonal Ab (dilution 1∶500; R&D Systems, Minneapolis, MN), and anti-GAPDH mouse monoclonal Ab (dilution 1∶5000; Millipore, Billerica, MA).

Techniques: Knock-Out, Immunofluorescence, Microscopy, Control, Labeling

Journal: PLoS ONE

Article Title: Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis

doi: 10.1371/journal.pone.0054239

Figure Lengend Snippet: Summary of the observed regulation in response to the AMC serum in renal cortices with various MK2 and MK3 genotypes.

Article Snippet: The following primary antibodies (Ab) were used: anti-MK2 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling, Danvers, MA), anti-MK3 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-β-actin rabbit monoclonal Ab (dilution: 1∶5000; Cell Signaling), anti-eIF2α mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-phospho-eIF2α rabbit monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-CHOP mouse monoclonal Ab (dilution: 1∶1000; Cell Signaling), anti-HSPB1 rabbit polyclonal Ab (dilution: 1∶5000; Assay Designs), anti-phospho-HSPB1-Ser86 rabbit polyclonal Ab (dilution: 1∶1000; Cell Signaling), anti-GRP78 rabbit polyclonal Ab (dilution: 1∶1000; StressMarq, Victoria, BC), anti-Hsp70 mouse monoclonal Ab (dilution: 1∶1000; StressMarq), anti-HSPB8 mouse monoclonal Ab (dilution: 1∶1000; Abcam, Cambridge, MA), anti-HSPB6 rabbit polyclonal Ab (dilution: 1∶1000; Abcam), anti-MK5 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-MK5 rabbit polyclonal Ab (dilution 1∶500; Pierce Biotechnology, Rockford, IL), anti-MNK1 rabbit monoclonal Ab (dilution 1∶1000; Cell Signaling), anti-phospho-Mnk1 polyclonal Ab (dilution 1∶1000; Cell Signaling), anti-MSK1 rabbit polyclonal Ab (dilution 1∶500; LifeSpan BioSciences, Seattle, WA), anti-phospho-MSK1 rabbit polyclonal Ab (dilution 1∶500; R&D Systems, Minneapolis, MN), and anti-GAPDH mouse monoclonal Ab (dilution 1∶5000; Millipore, Billerica, MA).

Techniques: Phospho-proteomics

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: A) Immunoblot analysis of total cellular extracts of Neo, WT and R120G HeLa cells. The levels of HspB1, HspB5, Hsp70, Hsp90, HspB6 and Actin were detected in immunoblots probed with the corresponding antibodies (see ). B) Phase contrast analysis of Neo, WT and R120G. bar: 10 µm. Black arrows: perinuclear granules; white arrowheads: membranous ruffles. Analysis of the biggest dimension of cells (cell length) is presented in the adjacent figure. Mean, SD (standard deviation) and SEM (standard error of mean) of twenty different measurements are presented. C) Analysis of the number of cells in the cultures was from day 0 to days 1 and 2 (d1, d2). Values are means ± SEM of three independent experiments. One-way ANOVA within a time point analysis indicates statistically significant growth differences between Neo and WT and R120G cell lines, *P <0.05. D) Immunofluorescence analysis. Neo, WT and R120G cells were processed for the immunofluorescence detection of HspB5, HspB1 and nuclei as described in . Bar: 10 µm. Cells were stained for HspB5 (red fluorescence), HspB1 (green fluorescence), nuclei (blue fluorescence) and processed as described in . The fusion images (Merge) of WT and R20G cells are shown. Overlap and Pearson's coefficients are indicated. E) The graphs represent the fluorescence distribution of HspB1 (green; Ch1-1), wild type or mutant HspB5 (red; Ch1-2) and nucleus (blue; Ch1-3) of the section of WT or R120G cells shown in the green/red fusion images (Merge). #: areas where the co-localization of HspB1 and HspB5 (wild type or mutant) may not occur.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Western Blot, Standard Deviation, Immunofluorescence, Staining, Fluorescence, Mutagenesis

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: A) Cellular distribution of HspB1, HspB5 (wild type and mutant) and Hsp70 upon cell lysis. Neo, WT and R120G cells were lysed in the presence of 0.1% Triton X-100 and spun at 10,000× g as described in . The levels of HspB1, HspB5 and Hsp70 present in the supernatant and pellet fractions were detected in immunoblots probed with the corresponding antibodies (see ). Autoradiographs of ECL-revealed immunoblots are presented. Quantitative analysis of three independent experiments is presented in the adjacent figure. B) Effect of shRNA-mediated depletion of HspB1. WT and R120G cells were transiently transfected with control mismatch pSuperNeo-MsRNA27 (Mismatch: Ms) or pSuperNeo-ShRNA27 (ShB1) vector targeting HspB1 mRNA (see ). Two days after transfection, cells were analyzed in immunoblots probed with HspB1, HspB5 and actin antibodies. ShB1 transfected cells were also treated for the last 20 h before being analyzed with 0.5 µmol/l of MG132. Quantitative analysis of one particular experiment where the RNAi-mediated transient depletion of HspB1 was almost complete is presented in the adjacent figure. C) Analysis of HspB1 and HspB5 native sizes in Neo, WT and R120G cells. Cells were lysed as above and the 10,000× g cytosolic supernatant fractions were applied to Sepharose 6B gel filtration columns (see ). The presence of HspB1 and HspB5 in pooled fractions eluted from the columns was detected in immunoblots probed with the corresponding antibodies. Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 kDa are gel filtration markers. Exclusion size of the column is 2000 kDa. Brackets indicate fractions that were pooled for further immunoprecipitation analysis. Size population I is from WT cells and size population II is from R120G cells. D) Co-immunoprecipitation studies. a) Size population I from WT cells was immunoprecipitated with either anti-HspB1 (IPαB1) or anti-HspB5 antibody (IPαB5). Immunoprecipitated proteins-bound to proteinG-sepharose were washed in IPP150 buffer containing 150 mM NaCl before being processed for gel electrophoresis. After migration in SDS-PAGE, proteins were revealed in immunoblots probed with either anti-HspB1 or anti-HspB5 antibody. T: aliquot of cytosolic supernatant fractions before immunoprecipitation, IP: immunoprecipitated proteins, S: aliquot from supernatant after immunoprecipitation. b) Same as a) except that washes of the immunoprecipitated proteins were performed in IPP300 buffer containing 300 mM NaCl. c–d) same as a–b) but in this case analysis was performed with size population II from R120G cells. Autoradiographs of ECL-revealed immunoblots are presented.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Mutagenesis, Lysis, Western Blot, shRNA, Transfection, Control, Plasmid Preparation, Filtration, Immunoprecipitation, Nucleic Acid Electrophoresis, Migration, SDS Page

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: Neo, WT or R120G cells were treated or not for different time periods with several concentrations of menadione. A) Crystal violet staining. The percentage of cell survival corresponded to the ratio of the relative absorbance of the different samples to that of untreated cells. Values are means ± SDM of three independent experiments. 2-way ANOVA indicates statistically significant differences in the survival to menadione treatment between Neo, WT and R120G cell lines, *P <0.05, **P <0.01. B) Clonogenic colony formation assay. The number of colonies was visually estimated. All experiments were performed in triplicate. C) Phase-contrast analysis of cell morphology. Before and after treatments, phase contrast analysis of the morphology of live cells was performed and photographs are presented. Bar: 10 µm. Black arrows: perinuclear granules; black arrowheads: filamentous bridges between cells; white arrowheads: membranous ruffles; white arrows: vacuoles. D) Immunoblot analysis of the level of HspB1, HspB5, Hsp70 and Hsp90 in menadione-treated Neo, WT and R120G cells.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Staining, Colony Assay, Western Blot

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: A) Immunoblot analysis of total cellular proteins. HeLa cells were either kept untreated (NT) or treated for 2 h with 60 µM menadione. Total cellular protein extracts were analyzed in immunoblots probed with antibodies that are specific for HspB1 or HspB5 and for HspB1 phosphorylation at either serine 15 (Ser15), 78 (Ser78) or 82 (Ser 82) or HspB5 phosphorylation at either serine 19 (Ser19), 45 (Ser45) or 59 (Ser59) (see ). The corresponding levels of total HspB1, HspB5 and actin are shown as controls (marked by a dark line surrounding immunoblots). Autoradiographs of ECL-revealed immunoblots are presented. B) Quantitative analysis of HspB1 and HspB5 phosphorylation in untreated cells and following exposure to menadione. Ba) Level of HspB1 and HspB5 phosphorylation in the immunoblot presented in A. The level of actin was used as standard of equivalent protein loading. Bb) Modulation of the phosphorylation of HspB1 and HspB5 by the R120G mutation. The R120G/WT ratio of the different HspB1 and HspB5 phosphoserine sites was defined as the ratio between the level of phosphorylation in R120G cells to that observed in WT cells. The R120G/WT ratios were calculated from three independent experiments. NT: non-treated. Treated: menadione-treated. Standard deviations are indicated (n = 3). Note the positive effect of the mutation towards HspB5 phosphorylation. C) Immunoblot analysis of fractionated cells. Same as A) but in this case 10,000× g supernatant (S) and pellet (P) fractions were analyzed from cells lysed in TEM buffer containing 0.1% Triton X-100 and spun at 10,000 g for 10 min. As in A, the corresponding levels of total HspB1, HspB5 and actin are shown as controls (marked by a dark line surrounding immunoblots). Autoradiographs of ECL-revealed immunoblots are presented. D) Quantitative analysis. The phosphorylation index of pellet fraction was defined as the ratio of the percentage of the phosphorylated protein in pellet to the percentage of the protein in that particular fraction. A value of 1.0 indicates that phosphorylation is proportional to the level of the protein in the pellet fraction. A value>1.0 is indicative of an enhanced phosphorylation of the protein in the insoluble fraction. A value<1.0 is indicative of a decreased phosphorylation of the protein in the insoluble fraction. Standard deviations are indicated (n = 3). Note the decreased phosphorylation of mutant HspB5 in the pellet of untreated R120G cells and the stimulated phosphorylation of pelleted wild type HspB5 in response to oxidative stress.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Western Blot, Phospho-proteomics, Mutagenesis

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: Non-treated as well as menadione-treated (60 µM, 2 h) Neo, WT and R120G cells were lysed and the S10,000× g supernatant fractions were applied to Sepharose CL-6B gel filtration columns as described in . The presence of HspB1, HspB5 and their different phosphorylated isoforms (HspB1: Ser15, Ser78 and Ser82/HspB5: Ser19, Ser45 and Ser59) were detected in immunoblots of the collected fractions probed with the corresponding antibodies. The corresponding native size of total HspB1 or HspB5 is shown (marked by a dark line surrounding immunoblots). Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 are gel filtration markers. Exclusion size of the column is 2000 kD. Quantitative analysis of the presence of HspB1 and HspB5 in different size domains of the column is presented (Neo cells: a, b, c and d; WT cells: e, f and g; R120G cells: h, i and j). Results are presented as percentage of HspB1, HspB5 and their different phosphorylated isoforms in the different size populations in regard to their amount in the S10,000× g supernatants. Standard deviations are indicated from three independent experiments. **P <0.01.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Filtration, Western Blot

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: The data presented in , and were used to calculate the percentage in regard to the total cellular content of HspB1, HspB5 and their different phosphorylated isoforms (HspB1: Ser15, Ser78 and Ser82/HspB5: Ser19, Ser45 and Ser59) in the different size populations (S10,000× g , gel filtration) and pellet fractions (P10,000× g ). Size populations from gel filtration columns and color codes are the same as those described in . Standard deviations are indicated from three independent experiments. **P <0.01.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Filtration

Journal: PLoS ONE

Article Title: Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

doi: 10.1371/journal.pone.0070545

Figure Lengend Snippet: A) Neo, WT and R120G cells were transiently transfected with pKS27S15G vector (phosphoserine 15 of HspB1 replaced by glycine, S15G vector; see ) and HspB1 as well as HspB1/HspB5 native sizes were subsequently analyzed as described in . Results obtained from R120G cells transiently transfected with the corresponding empty vector are also presented (control vector). Three size domains (I, II, and III) were defined in the R120G column fractions that corresponded to the h, i and j fractions indicated in and . Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 are gel filtration markers. Exclusion size of the column is 2000 kD. B) Quantitative analysis. The distribution of HspB1 between size domains I, II and III of transiently transfected R120G cells is shown and expressed as percentage of the HspB1 content in the 10,000× g supernatant loaded on the column. The following vectors were used: pKS control, pKS27S15G (phosphoserine 15 of HspB1 replaced by glycine), pKS2711-3D (the three phosphoserine sites of HspB1 replaced by aspartic acid) and pKS27wt (wild type HspB1). Standard deviations are indicated from three independent experiments.

Article Snippet: Recombinant HspB1 and HspB5 were from Enzo-Covalab (Villeurbanne, France).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Filtration

Journal: Technology in Cancer Research & Treatment

Article Title: Identification and Comparison of Differentiation-Related Proteins in Hepatocellular Carcinoma Tissues by Proteomics

doi: 10.1177/1533034617732426

Figure Lengend Snippet: Proteins Identified by MALDI-TOF-MS. a

Article Snippet: Rabbit anti-ANXA2 (Cat. No: YT0236), anti-HSPB1 (Cat. No: YM3356), anti-peroxiredoxin-2 (PRDX2; Cat. No: YT1706), and anti-calreticulin (CALR; Cat. No: YT0620)—all obtained from ImmunoWay Biotechnology Company (Plano, Texas)—were diluted at 1:300.

Techniques: Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: Identification and Comparison of Differentiation-Related Proteins in Hepatocellular Carcinoma Tissues by Proteomics

doi: 10.1177/1533034617732426

Figure Lengend Snippet: H&E staining demonstrating the well-differentiated HCC (A) and poorly differentiated HCC (B). Immunohistochemistry for ANXA2 in poorly differentiated HCC (C), ANXA2 in well-differentiated HCC (D), PRDX2 in poorly differentiated HCC (E), PRDX2 in well-differentiated HCC (F). Heat shock protein β-1 in poorly differentiated HCC (G), HSPB1 in well-differentiated HCC (H), CALR in poorly differentiated HCC (I), and CALR in well-differentiated HCC (J). ANXA2, annexin A2; CALR, calreticulin; HCC, hepatocellular carcinoma; HSPB1, heat shock protein β-1; PRDX2, peroxiredoxin-2.

Article Snippet: Rabbit anti-ANXA2 (Cat. No: YT0236), anti-HSPB1 (Cat. No: YM3356), anti-peroxiredoxin-2 (PRDX2; Cat. No: YT1706), and anti-calreticulin (CALR; Cat. No: YT0620)—all obtained from ImmunoWay Biotechnology Company (Plano, Texas)—were diluted at 1:300.

Techniques: Staining, Immunohistochemistry