hsp70 inhibitor ver 155008  (MedChemExpress)

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70s Regulate Circadian Rhythm under various conditions. A Immunoprecipitation assays were performed to compare the interaction strength between wild-type PER and Hsp70Ba under various temperature conditions. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and rabbit anti-Actin (ABclonal, #AC026). B Statistics of ( A ). N = 3. Statistical differences were assessed using one-way ANOVAs and post-hoc Tukey tests (P < 0.05). C Representative double plot actograms showing average locomotor activity for the respective genotypes under heat shock conditions. White background indicates light, and black indicates darkness. D Analysis of circadian rhythm persistence in Hsp70 mutants and w 1118 controls following heat shock. P-values from two-sided Fisher’s exact test are indicated (***P < 0.001)

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Immunoprecipitation, Western Blot, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Hsp70s regulate circadian rhythm by interacting with PERIOD

doi: 10.1186/s12964-026-02805-3

Figure Lengend Snippet: Hsp70 Protein Interacts with PER, Affecting PER Phosphorylation and Cellular Localization. A Relative per expression at different time points in Hsp70 mutants and the control group. B Antibody specificity evaluation in western blot analysis for PER protein. C PER protein detection by western blots at ZT3, ZT9, ZT15, and ZT21. B - C Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and mouse anti-β-Tubulin (ABclonal, Cat#AC021). D Quantification of results in ( C ). N = 5. Statistical differences were measured using an unpaired Student’s t-test. **P < 0.01, ***P < 0.001. E Statistics of the cellular localization of PER in s-LNvs and l-LNvs clusters of clock neurons in indicated genotypes. C represents cytoplasm, and N represents nucleus. F Immunoprecipitation experiments to detect the interaction between PER, TIM, and Hsp70s. Western blot analysis was performed with mouse anti-V5 (ABclonal, Cat#AE017), mouse anti-HA (ABclonal, Cat#AE008) antibodies and mouse anti-β-Tubulin (ABclonal, Cat#AC021). Samples were prepared in S2 cells, with β-tubulin used as an internal control. G Calculation of average TIM binding by PER molecule. N = 3. Significance of differences was determined using one-way ANOVAs and post-hoc Tukey tests. H Immunoprecipitation experiments to detect the interaction between PER and Hsp70s in tim 01 and w 1118 controls. Western blot analysis was performed with anti-PER (a gift from Dr. Jeffrey Price’s laboratory at the University of Missouri-Kansas City, U. S.) and anti-β-Tubulin (ABclonal, Cat#AC021). β-tubulin was used as an internal control. ( I ) Calculation of average Hsp70 binding by PER molecule. N = 5. The t-test was used to calculate the significance of differences. **P < 0.01

Article Snippet: For drug treatment, 100 μM of the Hsp70 inhibitor VER-155,008 (Target Mol, #T7010) was dissolved in the media.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Immunoprecipitation, Binding Assay

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: Intermittent heat stimulation training improves high temperature tolerance in mice. A. Flowchart of the experiment to improve heat tolerance in mice through intermittent heat stimulation training. B & C. ELISA was used to analyze the plasma concentrations of cytokines (IL-1β and TNF-ɑ) in control, HS, and HS+HA group mice (n =8) 16 h after HS. D. WB was used to detect the expression of HIF-1α and HSP70 protein. β-actin was used as an internal control. E. IF analysis of isolated microvessels stained with antibodies for each cell component. Endothelial markers (CD31, green channel; VWF, red channel) were detected in all microvessel fragments. Scale bar = 50 μm. F. IF was used to detect apoptosis in isolated microvessels stained with TUNEL. Scale bar = 100 μm. **** P < 0.0001.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Expressing, Isolation, Staining, TUNEL Assay

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA induces tolerance to high temperatures via the HIF-1α/HSP70 signaling. A & B. The bEnd.3 cells were exposed to 40℃ for 2 h daily, continuously for 1 day, 3 days, and 5 days. RT-qPCR was used to detect the expression of Hif-1α and Hspa1a mRNA. C. WB was used to detect the expression of HIF-1α, HSP90AA1, and HSP70 protein. β-actin was used as an internal control. D. IF was used to detect the expression of HIF-1α and HSP70 protein. E. WB was used to detect the expression of HIF-1α and HSP70 protein. β-actin was used as an internal control. F. bEnd.3 HA cells were exposed to HS for 4 h and treated with LW6, VER-155088, or a combination of both. Cell viability was detected using the CCK-8 assay. G. bEnd.3 HA cells were exposed to HS for 4 h and treated with si- Hif-1α , si- Hspa1a , or a combination of both. Cell viability was detected using the CCK-8 assay. H. Flow cytometry with Annexin V and PI staining was used to determine the apoptotic index of cells. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA enhances HIF-1α protein stability via HSP70 in bEnd.3 cells. A. bEnd.3 and bEnd.3 HA cells were treated with CHX for the indicated times and analyzed by WB. B. IF analysis of HIF-1α subcellular localization. Nuclei are stained with DAPI (blue). n = 5 per group, scar bar = 50 μm. C. bEnd.3 HA cells were treated with DMSO, si-NC, VER-155008, or si- Hspa1a and with CHX for 6 h. WB was used to detect the HIF-1α protein expression. D. IF was used to detect the HIF-1α protein expression. Nuclei are stained with DAPI (blue). n=5 per group, scar bar = 50 μm. E. bEnd.3 and bEnd.3 HA cells were treated with si-NC, VER-155008, or si- Hspa1a . The extracts were immunoprecipitated with anti-HIF-1α antibodies and immunoblotted with anti-ubiquitin, anti-HIF-1α, and anti-HSP70 antibodies. β-actin was used as an internal control.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Staining, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Control

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA promotes the interaction between HIF-1α and HSP70 proteins in bEnd.3 cells. A. ZDOCK prediction was used to model the predicted interaction between HSP70 and HIF-1α. B. Predicted ZDOCK binding score, hydrogen bonds, and electrostatic interactions with amino acid sites between HSP70 and HIF-1α. C. Colocalization of HIF-1α (red) with HSP70 (green) in bEnd.3 and bEnd.3HA cells. Nuclei are stained with DAPI (blue). n=5 per group, scar bar = 50 μm. D & E. bEnd.3 and bEnd.3HA cells were treated with si-NC, si-Hif-1α, or si-Hspa1a. The extracts were immunoprecipitated with anti-HIF-1α or anti-HSP70 antibodies and immunoblotted with anti-HIF-1α and anti-HSP70 antibodies.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Binding Assay, Staining, Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: HA induces tolerance to hypoxia via the HIF-1α/HSP70 signaling. A. bEnd.3 and bEnd.3 HA cells were treated at 1% O 2 concentration for 12 h. Cell viability was detected using the CCK-8 assay. B. Flow cytometry with Annexin V and PI staining was used to determine the apoptotic index of cells. C. WB was used to detect the expression of p53, Bcl-2, Bax, and cleaved caspase3 protein. β-actin was used as an internal control. D. bEnd.3 HA cells were treated at 1% O 2 concentration for 12 h and with LW6, VER-155008, or LW6+VER-155008. Cell viability was detected using the CCK-8 assay. E. bEnd.3 HA cells were treated at 1% O 2 concentration for 12 h and with si- Hif-1α , si- Hspa1a , or si- Hif-1α +si- Hspa1a . Cell viability was detected using the CCK-8 assay. F. Flow cytometry with Annexin V and PI staining was used to determine the apoptotic index of cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Concentration Assay, CCK-8 Assay, Flow Cytometry, Staining, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Heat acclimation mediates cellular protection via HSP70 stabilization of HIF-1α protein in extreme environments

doi: 10.7150/ijbs.103122

Figure Lengend Snippet: Diagram representing the potential mechanism by which HA regulates stabilization of HIF-1α protein via HSP70 in bEnd.3 cells. Under normal conditions, HIF-1α protein is ubiquitinated and degraded by the proteasome. Under HA conditions, HSP70 interacts with the HIF-1α protein, inhibiting its ubiquitination, stabilizing the HIF-1α protein, and promoting the transcription of downstream genes in the nucleus.

Article Snippet: BioTracker ERthermAC (SCT057, Merck, Darmstadt, Germany), HSP70 inhibitor (VER-155008, HY-10941, MCE, New Jersey, USA), HIF-1α inhibitor (LW6, HY-13671, MCE), Cycloheximide (CHX, HY-12320, MCE), Vari Fluor 488-Phalloidin (HY-D1817, MCE), Primary antibodies: anti-β-Actin (1:10000, 60008-1-Ig, Proteintech, Illinois, USA), anti-Ubiquitin (1:3000, 10201-2-AP, Proteintech), anti-P53 (1:3000, 60283-2-Ig, Proteintech), anti-Bcl2 (1:3000, 26593-1-AP, Proteintech), anti-Bax (1:2000, 60267-1-Ig, Proteintech), anti-Cleaved-Caspase3 (1:3000, 25128-1-AP, Proteintech), anti-HIF-1α (1:3000, #9542, CST, USA), anti-HSP70 (1:5000, CY5496, Abways, Shanghai, China), anti-HSP90 (1:5000, BM4387, BOSTER, California, USA), Hematoxylin & Eosin stain (HE, C0105S, Beijing, China), Nissl stain (C0117, Beyotime), Matrigel (Corning, 354234, New York, USA), Cell counting kit-8 (CCK-8, K1018, APExBIO Houston, USA), Dulbecco's Modified Eagle Medium-High glucose (DMEM, C11875500BT, Gbico, Waltham, USA), Fetal Bovine Serum (FBS, BS1612-109, Bioexplorer, Caesarea, Israel), Penicillin/streptomycin (P/S, BL505A, Biosharp, Beijing, China), ATP assay kit (S0026, Beyotime), TUNEL assay kit (C1088, Beyotime), BCA assay kit (P0010S, Beyotime), RIPA (P0038, Beyotime), Annexin V-FITC apoptosis detection kit (C1062M, Beyotime), Lipofectamine 3000 (L3000-008, Invitrogen, Waltham, USA), Evans blue (EB, E2129, Sigma-Aldrich, Missouri, USA).

Techniques: Ubiquitin Proteomics