hsp70 inhibitor ver  (MedChemExpress)

Journal: eLife

Article Title: p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners

doi: 10.7554/eLife.103537

Figure Lengend Snippet: ( A ) Solubility assay of p53 isoforms in new vector system (p5RPU.myc). H1299 cells were transiently transfected with the indicated Myc-tagged proteins and lysed in the respective buffers. Lysates were separated by centrifugation with the soluble components being in the supernatant (S) and the insoluble in the pellet (I). Samples were subsequently analyzed by WB using an α-Myc antibody. Vinculin served as loading control. ( B ) Solubility assay of Δ40p53α in the presence either DMSO or different inhibitors for HSP70 (JG-98, YM-1, VER-155008) or HSP90 (17-AAG) showing that these inhibitors have virtually no effect on the solubility of this isoform. ( C ) and ( D ) Same assay as in ( E ) but with the isoforms ΔN133p53α ( F ) and Δ133p53β ( G ) demonstrating that the use of chaperone inhibitors increases the insoluble fraction. Figure 4—figure supplement 3—source data 1. Uncropped Western blots.

Article Snippet: The used inhibitors were JG-98 (HSP70 inhibitor, final concentration 5 μM, MedChem Express), YM-1 (HSP70 inhibitor, final concentration 10 μM, MedChem Express), VER-155008 (HSP70 inhibitor, final concentration 10 μM, MedChem Express), and 17-AAG (HSP90 inhibitor, final concentration 2 μM, MedChem Express).

Techniques: Solubility, Plasmid Preparation, Transfection, Centrifugation, Control, Western Blot

Journal: eLife

Article Title: p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners

doi: 10.7554/eLife.103537

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of p53 isoforms and cancer-related mutants with endogenous HSC/HSP70. H1299 cells were transiently transfected with either empty vector or the indicated N-terminally Myc-tagged p53 variants. HSC/HSP70 was immunoprecipitated (IP) with an αHSC/HSP70 antibody. Input and IP samples were subsequently analysed by WB using αHSC/HSP70 and α-Myc antibody to detect HSC/HSP70 and p53 variants, respectively. A light and dark exposure of the IP samples detected with α-Myc antibody is shown. Vinculin served as a loading control for the input samples. ( B ) Luciferase reporter assay of p53 isoforms and mutants as well as the indicated Scarlet fusion proteins on the HSP70 promoter. H1299 cells were transiently transfected with the respective luciferase reporter plasmids and the N-terminally Myc-tagged proteins. ( B ) p53 (331) contains only the C-terminal part common to p53α, p53β, and p53γ. The fluorescent protein Scarlet was fused with the C-termini of p53 isoforms. Scarlet alone served as a negative control. ( C ) Luciferase reporter assay of p53 isoforms and mutants as well as the indicated Scarlet fusion proteins on the heat-shock element (HSE) promoter (containing three repeats of heat shock element). H1299 cells were transiently transfected with the respective luciferase reporter plasmids and the N-terminally Myc-tagged proteins. ( C ) p53 (331) contains only the C-terminal part common to p53α, p53β, and p53γ. The fluorescent protein Scarlet was fused with the C-termini of p53 isoforms. Scarlet alone served as a negative control. ( B, C ) The bar diagram shows the mean fold induction relative to the empty vector control and error bars the corresponding SD ( n = 3). Statistical significance was assessed by ordinary one-way ANOVA (n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). ( D ) Chaperones and other proteins associated with binding un-/misfolded proteins are shown, which are significantly enriched in the mass spectrometry analysis of p53 isoforms. The red nods represent the identified proteins assigned by the grey lines to the p53 isoforms they were enriched for. Significant hits were determined by setting the parameters: log 2 enrichment greater than or equal to 0.5 and p-value less than 0.05 and proteins were filtered for the keywords (‘chaperone’, ‘unfolded protein binding’, and ‘misfolded protein binding’) in the GOBP (Gene Ontology Biological Process) and GOMF (Gene Ontology Molecular Function) terms. The plot was generated using DiVenn (v2.0). ( E ) Results from the second mass spectrometry experiment, focused on the quantification of p53 peptide precursors. As the biotin ligase was directly fused to p53, p53 peptides are quantified for all samples (log 2 -transformed protein LFQ intensity). Negative controls represent data from unbiotinylated lysates (no biotin added). ( F ) Peptides from the DNA-binding domains spanning residues 249–267 were quantified for all samples (log 2 -scaled precursor intensity), consistent with this peptide being part of all investigated isoforms. A peptide spanning amino acids 102–110 was absent or beyond the detection limit in the Δ133p53β sample but quantified in all other samples. ( G ) Peptides originating from the oligomerization domain were reliably quantified in samples of isoforms containing a full-length oligomerization domain (log 2 -scaled precursor intensity), but not in samples of isoforms containing the β- and ɣ-C-termini (except for one low intense TAp53β precursor ion). Figure 5—source data 1. Uncropped Western blots.

Article Snippet: The used inhibitors were JG-98 (HSP70 inhibitor, final concentration 5 μM, MedChem Express), YM-1 (HSP70 inhibitor, final concentration 10 μM, MedChem Express), VER-155008 (HSP70 inhibitor, final concentration 10 μM, MedChem Express), and 17-AAG (HSP90 inhibitor, final concentration 2 μM, MedChem Express).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Control, Luciferase, Reporter Assay, Negative Control, Binding Assay, Mass Spectrometry, Protein Binding, Generated, Transformation Assay, Western Blot

Journal: eLife

Article Title: p53 isoforms have a high aggregation propensity, interact with chaperones and lack binding to p53 interaction partners

doi: 10.7554/eLife.103537

Figure Lengend Snippet: ( A ) Co-IP of p53 isoforms and cancer-related mutants. H1299 cells were transiently transfected with N-terminally Myc-tagged p53 variants. p53 was immunoprecipitated (IP) with either α-mouse IgG or α-HSP70 antibody. Input and IP samples were subsequently analyzed by WB using an α-Myc antibody. For the relative Co-IP efficiency each IP signal was normalized to the input signal. ( B ) Expression levels of the transiently transfected proteins in the luciferase reporter assay from were determined by WB using an α-Myc antibody. Vinculin served as loading control. p53 (331) contains only the C-terminal part common to p53α, p53β and p53γ. ( C ) Expression levels of the transiently transfected proteins in the luciferase reporter assay from were determined by WB using an α-Myc antibody. Vinculin served as loading control. p53β CT: aa 332-341; p53γ CT: aa 332-346; p53γ CT mut: aa 332-346 with Y327A L330A L334A L336A; p53ψ CT: aa 223-243. Figure 5—figure supplement 1—source data 1. Uncropped Western blots.

Article Snippet: The used inhibitors were JG-98 (HSP70 inhibitor, final concentration 5 μM, MedChem Express), YM-1 (HSP70 inhibitor, final concentration 10 μM, MedChem Express), VER-155008 (HSP70 inhibitor, final concentration 10 μM, MedChem Express), and 17-AAG (HSP90 inhibitor, final concentration 2 μM, MedChem Express).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Expressing, Luciferase, Reporter Assay, Control, Western Blot

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Benzene metabolites increase vascular permeability by activating heat shock proteins and Rho GTPases.

doi: 10.1093/toxsci/kfaf055

Figure Lengend Snippet: Fig. 3. Muconaldehyde-induced stimulation of HSPs in endothelial cells. (A) MCMVEC were incubated with 10 μM MA for 6 h and gene transcription was measured by RNA sequencing. (i) Volcano plot of differentially expressed genes. (ii) Reactome pathway enrichment analysis of MA-treated MCMVEC. (B) HAECs were incubated with MA (10 µM) for 2, 6, or 24 h, and gene transcription was measured by RNA sequencing. (i) Venn diagram of differentially expressed genes at indicated time points (padj < 0.05; log2FoldChange ≥j2j). (ii) Pathway enrichment analysis of MA-treated HAEC. (iii) HSP70 and HSP90 induction in MA-treated HAEC. (iv) Gene network of HSP-related pathways activated after 6 h exposure to MA. (C) MA (10 µM)-induced expression of Hspa1b gene (i) and upregulation of HSPA1B protein in MCMVEC (ii). (D) Immunofluorescent staining of Hsp70 and in MCMVEC treated with MA (10 µM) for 24 h. (E) Colocalization of Hsp70 with F-actin in MA-treated MCMVEC.

Article Snippet: HSP70 inhibitor JG98, HSP90 inhibitor 17-AAG, and ROCK1 (p160ROCK) inhibitor Y-27632 were purchased from the Selleckchem.com.

Techniques: Incubation, RNA Sequencing, Expressing, Staining

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Benzene metabolites increase vascular permeability by activating heat shock proteins and Rho GTPases.

doi: 10.1093/toxsci/kfaf055

Figure Lengend Snippet: Fig. 4. Contribution of HSPa in MA-induced changes in endothelial permeability. The endothelial monolayer was exposed to a medium containing 0.1% DMSO in PBS or 10 µM MA for the indicated times. Fluorescent dextran concentrations were measured in the lower chambers as described in the Materials and Methods section. Cells were co-exposed to the HSP90 inhibitor 17-AAG (A, 2 μM) or the HSP70 inhibitor JG98 (C, 2 μM). Data are shown as mean±SE. P < 0.05, P < 0.01, P < 0.0001 for MA versus Control; # #P < 0.01, # # # #P < 0.0001 for inhibitor/activator/MA versus MA, 2-way ANOVA with Tukey posttest. The AUC was calculated for the effects of 17-AAG (B) and JG98 (D) on MA-induced permeability. Data are shown as mean±SE. P < 0.05, P < 0.01, P < 0.001, P < 0.0001, 1-way ANOVA with Bonferroni posttest. (E) Characterization of HSPA1B-TGEC mice. (i) Schematic of transgene construct used for HSPA1B-TGEC mice generation. Mice that were homozygous for Hspa1b were crossed with mice expressing Cre recombinase under the control of the endothelial-specific Cdh5 promoter. (ii) Genotyping of transgenic mice. (iii) Detection of HSPA1b levels in isolated lung endothelial cells (EC) using western blot. (iv) The Hspa1bþ/þ/Creþ/− (HSPA1B-TGEC) and the corresponding littermate controls (Hspa1bþ/þ/Cre−/−) mice were injected (i.v.) with Evans blue and then treated with intradermal injections of MA (400 pmol per injection) or the vehicle. Group data on vascular leakage in skin extracts 30 min after MA treatment. Data are mean±SEM. P < 0.05, P < 0.001,P < 0.0001 following 2-way ANOVA with Tukey posttest. (v) MA-induced fold change in the vascular leakage detected in HSPA1B-TGEC versus littermate controls. Data are mean±SEM. P < 0.05 as per 2-tailed t-test (unpaired).

Article Snippet: HSP70 inhibitor JG98, HSP90 inhibitor 17-AAG, and ROCK1 (p160ROCK) inhibitor Y-27632 were purchased from the Selleckchem.com.

Techniques: Permeability, Control, Construct, Expressing, Transgenic Assay, Isolation, Western Blot, Injection

Journal: Cell reports

Article Title: Geranylgeranylated SCF FBXO10 regulates selective outer mitochondrial membrane proteostasis and function

doi: 10.1016/j.celrep.2024.114783

Figure Lengend Snippet:

Article Snippet: HSP70 Inhibitor, PES-Cl , Millipore Sigma , Cat#5.31067.

Techniques: Recombinant, Protease Inhibitor, Sequencing, Retroviral, Quantitative Proteomics, Mass Spectrometry, Western Blot, Software