h90 10  (Developmental Studies Hybridoma Bank)

Journal: Journal of visualized experiments : JoVE

Article Title: Mapping Dysfunctional Protein-Protein Interactions in Disease

doi: 10.3791/69197

Figure Lengend Snippet: ( A ) Biological specificity and lot-to-lot consistency. MDA-MB-468 (epichaperome-high) and ASPC1 (epichaperome-low) cells were lysed in native buffer (20 mM Tris, pH 7.4; 20 mM KCl; 5 mM MgCl 2 ; 0.01% NP-40; protease/phosphatase inhibitors). For each capture, 40 μL of PU-bead slurry was incubated with 250 μg of total protein (1 μg/μL) for 3 h at 4 °C with rotation. Beads were washed in native buffer; complexes were denatured/eluted in ~100 μL of SDS sample buffer. 5-10 μL of each eluate was resolved by SDS-PAGE and immunoblotted for HSP90, HSC70, and HOP. Input lysates are shown for reference. Two PU-bead lots (Batch 1, fresh; Batch 2, aged) yield strong enrichment in MDA-MB-468 and minimal signal in ASPC1, demonstrating preserved specificity and lot consistency. ( B ) Global cargo versus control beads. MDA-MB-468 lysates were processed as in (A) with either PU-beads or matched control beads. ~20 μL of each eluate was loaded, separated by SDS-PAGE, and Coomassie-stained. PU-beads recover a complex, high-MW cargo characteristic of epichaperome-bound assemblies, whereas control beads show minimal background. Molecular-weight markers (kDa) are indicated. Please click here to view a larger version of this figure .

Article Snippet: HSP90 (H90-10) antibody (RRID: AB_854214) , StressMarq , SMC-107 , For PU-beads QC.

Techniques: Incubation, SDS Page, Control, Staining, Molecular Weight

Journal: Current Research in Insect Science

Article Title: Isolation of extracellular vesicles from insect hemolymph

doi: 10.1016/j.cris.2025.100118

Figure Lengend Snippet: Characterization of S2-cell derived EVs. (A) Western blot analysis of the proteins ROP (67 kDa), Rab11 (24 kDa), Rho-1 (21 kDa), HSP90 (90 kDa) and Cnx (68 KDa), in the S2 cell-derived extracellular fractions isolated based on density with ODG ultracentrifugation. Left lanes correspond to the ladder and the ladder band size is indicated in kDa. The measured density of the fractions is indicated on the top, in g/mL. The fraction numbers are indicated on the bottom (1–12). Next, the EV-containing fractions (7–10) were pelleted by ultracentrifugation. (B) EV size assessed by NTA. The size distribution is depicted as a mean (black line) with standard error of the mean (red error bars). (C) EV morphology examined by TEM. Scale bar represents 200 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: Primary antibodies against the following proteins were used: Rab11 (610656, BD BioSciences), ROP (DSHB Hybridoma Product ROP 4F8, Rubin G.M.), Rho1 (DSHB Hybridoma Product p1D9 (anti-rho1), Parkhurst S.), Heat Shock Protein 90 (HSP90) (DSHB Hybridoma Product H90–10, Freeman B.), Calnexin (Cnx) (DSHB Product Cnx99A 6–2–1, Munro S.), α-Tub (DSHB Hybridoma Product 12G10 anti-alpha-tubulin, Frankel J., Nelsen E.M.), L. migratoria Argonaute-1 and Apolipophorin-I.

Techniques: Derivative Assay, Western Blot, Isolation

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: Hsp90 glutathionylation mainly occurred at C366 and C412 under oxidative stress . (A) MDA-MB-157 breast cancer cells were treated with or without H 2 O 2 (1 mM) for 10 min. The clarified lysates were subjected to immunoprecipitation (IP) with anti-Hsp90β antibody and western blot analysis with anti-glutathione (GSH) antibody to detect glutathionylation (PSSG-Hsp90β). The level of PSSG-Hsp90β was quantified and normalized to immunoprecipitates of His-Hsp90β (n = 4). * P < 0.05, compared to cells without H 2 O 2 treatment by student's t -test. (B) Glutathionylation levels of His-Hsp90β recombinant proteins (Hsp90β-SSG) incubated with various concentrations of oxidized glutathione (GSSG) were observed by western blot analysis with anti-GSH antibody. D , Dithiothreitol, as a reducing reagent. The signal of glutathionylation of His-Hsp90β (Hsp90β-SSG) was quantified and measured by the normalization of Hsp90β-SSG to total His-Hsp90β protein (Hsp90β-SSG/His-Hsp90β) (n = 3). * P < 0.05, versus to His-Hsp90β recombinant protein incubated with 10 mM GSSG by One-way ANOVA. (C) MDA-MB-157 cells were transfected with wild-type His-Hsp90β vector (WT), or vectors expressing His-Hsp90β with mutations of C366 (C366A), 412 (C412A), 521 (C521A) or 589 (C589A). After treatment with 1 mM H 2 O 2 for 10 min, an immunoprecipitation assay was performed with anti-His antibody, and glutathionylation was detected by anti-GSH antibody. Glutathionylated signal (GSH) was measured and normalized to His-precipitates (PSSG-Hsp90β/His-Hsp90β) (n = 3). * p < 0.05, compared to WT His-Hsp90β overexpressing cells without H 2 O 2 stimulation. # p < 0.05, compared to WT His-Hsp90β overexpressing cells with H 2 O 2 treatment by One-way ANOVA. (D) Purified WT or mutated forms of His-Hsp90β proteins were treated with 2 mM GSSG for 30 min, and the glutathionylation signal (GSH) was detected by western blot with an anti-GSH antibody. D, Dithiothreitol, as a reducing reagent. The level of glutathionylated His-Hsp90β protein was measured by normalization to His-Hsp90β protein (Hsp90β-SSG/His-Hsp90β) (n = 3). * P < 0.05, versus to WT Hsp90β protein incubated with 2 mM GSSG by One-way ANOVA. All data are shown as the mean (±SD).

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Immunoprecipitation, Western Blot, Recombinant, Incubation, Transfection, Plasmid Preparation, Expressing, Purification

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: Glutathionylation inhibited the ATPase activity of Hsp90 . Recombinant Hsp90β protein (2 μg) was incubated without or with oxidized glutathione (GSSG, at 2, 5 or 10 mM) for 30 min before an ATPase activity assay was performed. ATPase activity assay was conducted according to the protocol as described in materials and methods. The ATPase activity of Hsp90β protein without GSSG treatment was presented as 100%. ATPase activity of Hsp90β protein treated with GSSG was normalized to that of Hsp90β protein without GSSG treatment. D: Dithiothreitol. * p < 0.05, versus Hsp90β protein without GSSG treatment. # p < 0.05, compared to Hsp90β protein with 10 mM GSSG stimulation by One-way ANOVA. Results are presented as the mean percentage (±SD) of three independent experiments.

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Activity Assay, Recombinant, Incubation

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: H 2 O 2 -induced oxidative stress enhanced Hsp90 protein degradation . (A) WT His-Hsp90β or its corresponding mutant at the indicated cysteine (C366A, C412A, C521A, and C589A) was overexpressed ectopically in MDA-MB-157 cells that were further treated with or without 1 mM H 2 O 2 for 30 min. The expression of His-Hsp90β was examined and quantified by western blotting with anti-His antibody (n = 3). * p < 0.05, compared to that WT His-Hsp90β overexpressing cells without H 2 O 2 treatment. # p < 0.05, versus wild-type His-Hsp90β overexpressing cells stimulated with H 2 O 2 . (B) MDA-MB-157 cells carrying WT or a cysteine mutation at position 366, 412, 521 or 589 in His-Hsp90β were treated with 100 μg/ml cycloheximide (CHX) and 1 mM H 2 O 2 at the indicated time points. Western blot assay was performed to detect the expression levels of His-Hsp90β using anti-His antibody. (C) Relative expression levels of His-Hsp90β shown in (B) are plotted and quantified by normalization to GAPDH level (n = 3). * p < 0.05, compared to that WT His-Hsp90β overexpressing cells without H 2 O 2 treatment. # p < 0.05, compared to group of WT His-Hsp90β overexpressing cells stimulated with H 2 O 2 . Results are presented as the mean percentage (±SD) and analyzed by by One-way ANOVA.

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Mutagenesis, Expressing, Western Blot

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: S-glutathionylation promoted Hsp90 degradation through the increase of CHIP binding and ubiquitination . (A) Recombinant His-Hsp90β protein treated with or without 2 mM GSSG was incubated with cell extracts from MDA-MB-157 cells for the indicated times. MG-132 treatment (10 μM) was used to inhibit proteasome activity before His-Hsp90β protein exposure to GSSG. The expressional level of His-Hsp90β protein was detected by western blot analysis with anti-His antibody and normalized to level of GAPDH. The relative expression of His-Hsp90β protein at 0 min was represented as 100% of protein level (n = 3). * p < 0.05, compared to that WT His-Hsp90β protein without incubation with GSSG. # p < 0.05, compared to WT His-Hsp90β protein incubated with GSSG by One-way ANOVA. (B, C) WT His-Hsp90β protein or His-Hsp90β protein mutated at C366 (C366A) or C412 (C412A) was treated with or without 2 mM GSSG for 30 min. After incubation with cell extracts from MDA-MB-157 cells expressing HA-ubiquitin for 30 min, an immunoprecipitation (IP) assay was performed using an anti-His antibody. The ubiquitinated His-Hsp90β protein was detected by western blotting analysis with anti-HA antibody. The relative level of ubiquitinated His-Hsp90β protein was quantified by normalization the density of a smear in each individual lane to the density of His-Hsp90β protein (ubiquitinated His-Hsp90β/His-Hsp90β) in the precipitate. After immunoprecipitation with anti-His antibody, the binding with CHIP was analyzed using western blot with anti-CHIP antibody and quantified by normalization to the level of His-Hsp90β protein in the precipitates (n = 3). * p < 0.05, versus wild-type Hsp90β protein without GSSG treatment. # p < 0.05, compared to wild-type Hsp90β protein treated with 2 mM GSSG by One-way ANOVA. Data are shown as the mean (±SD).

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Binding Assay, Recombinant, Incubation, Activity Assay, Western Blot, Expressing, Immunoprecipitation

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: Hsp90 S-glutathionylation impaired CK2 recruitment and the subsequent C-terminal phosphorylation, leading to the increased CHIP binding . (A) Purified WT His-Hsp90β protein or His-Hsp90β mutated at S718 (S718D) was treated with or without 2 mM GSSG and then mixed with cell lysates from MDA-MB-157 cells for 30 min. Subsequent immunoprecipitation was performed with an anti-His antibody. Immunoprecipitated proteins were analyzed by western blot analysis with anti-CHIP antibody. The binding of CHIP to His-Hsp90β protein was measured by the expression of CHIP to the level of His-Hsp90β in the precipitates (n = 4). * p < 0.05, compared to WT His-Hsp90β protein without GSSG stimulation. # p < 0.05, compared to wild-type His-Hsp90β protein treated with 2 mM GSSG by One-way ANOVA. (B) His-Hsp90β protein incubated with 2 mM GSSG for 30 min was mixed with total cell protein from MDA-MB-157 cells for 30 min. Immunoprecipitation assay was performed by anti-His antibody. The interaction of CK2 and His-Hsp90β protein was further analyzed in the precipitates by western blot using anti-CK2 antibody. The binding of CK2 to His-Hsp90β protein was analyzed by the ratio of CK2/His-Hsp90β protein (n = 4). * p < 0.05, compared to His-Hsp90β protein without GSSG incubation. Results are presented as the mean percentage (±SD).

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Binding Assay, Purification, Immunoprecipitation, Western Blot, Expressing, Incubation

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: Secondary structure content of recombinant Hsp90β in non-glutathionylated or glutathionylated forms.

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Recombinant

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: The structure of Hsp90 was altered after S-glutathionylation . Recombinant His-Hsp90β protein (2 μg) was incubated without or with various concentrations of GSSG for 30 min. Far-UV CD spectra were measured to analyze the changes in secondary structures of His-Hsp90β protein.

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Recombinant, Incubation

Journal: Redox Biology

Article Title: S-glutathionylation of Hsp90 enhances its degradation and correlates with favorable prognosis of breast cancer

doi: 10.1016/j.redox.2022.102501

Figure Lengend Snippet: High expression of glutathionylated Hsp90 inversely correlated with a low level of total Hsp90 in breast cancer patients with a favorable prognosis . (A) Tumors from 64 breast cancer patients were lysed and homogenized to extract total cell lysates, which were subjected to immunoprecipitation assay using an anti-Hsp90β antibody. The level of glutathionylated Hsp90β (PSSG-Hsp90β) in Hsp90β-precipitates was detected by western blotting with anti-GSH antibody and normalized to level of Hsp90β in the precipitates. Expressional level of total Hsp90β (Hsp90β) in tumors was analyzed by western blotting with anti-Hsp90β antibody and normalized to level of GAPDH. Quantitative results for the levels of glutathionylated (PSSG-Hsp90β) and total Hsp90 (Hsp90β) were plotted. (B) The correlation between the levels of glutathionylated Hsp90β and total Hsp90β in primary breast cancer tumors was determined. The statistical analysis was performed using linear regression. (C – E) A box plot represented the ratio of total Hsp90β to glutathionylated Hsp90β in breast cancer tumors divided into ER-, PR-, and HER2-expressing subtypes.

Article Snippet: Anti-Hsp90β (H90-10) antibody was from Enzo Life Sciences. pThr725/Ser726 Hsp90α antibody (GDD8.2) was kindly provided by Dr. Vojtesek at Masaryk Memorial Cancer Institute, Czech Republic.

Techniques: Expressing, Immunoprecipitation, Western Blot