Journal: Scientific Reports

Article Title: Therapeutic effect of histone deacetylase 6 inhibitor for a mouse model of phenylketonuria

doi: 10.1038/s41598-025-29143-7

Figure Lengend Snippet: Western blot analysis of acetyl-HSP90, HSP90, and PAH protein expression in liver tissues from sham, treated, and wild-type groups following 12 weeks of TSA treatment. ( A ) Expression of acetyl-HSP90, HSP90, PAH, and GAPDH (used as a loading control). ( B ) Quantitative analysis of acetyl-HSP90 relative to HSP90 protein levels. ( C ) Quantitative analysis of PAH relative to GAPDH protein levels. The expression of PAE was quantified using the Image J software (Version 1.49) and expressed as arbitrary units (a.u.). Results are presented as mean ± SEM, with statistical significance indicated. ****P<0.0001.

Article Snippet: The antibodies used anti-acetylated HSP90 (600-401-981, Rockland Immunochemical, 1:500), anti-HSP90 (#4877, Cell Signaling, 1:1,000), and PAH (sc-271258, Santa Cruz, 1:2,000).

Techniques: Western Blot, Expressing, Control, Software

Journal: Scientific Reports

Article Title: Therapeutic effect of histone deacetylase 6 inhibitor for a mouse model of phenylketonuria

doi: 10.1038/s41598-025-29143-7

Figure Lengend Snippet: Mechanism of TSA in enhancing HSP90 acetylation and stabilizing misfolded PAH protein to improve phenylalanine metabolism.

Article Snippet: The antibodies used anti-acetylated HSP90 (600-401-981, Rockland Immunochemical, 1:500), anti-HSP90 (#4877, Cell Signaling, 1:1,000), and PAH (sc-271258, Santa Cruz, 1:2,000).

Techniques:

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 2. Hsp90 plays a pivotal role in the production of IFN-α by pDCs. (A, B) pDCs derived from HSF-1−/−mice showed decreased Hsp90α expression and impaired ability to produce IFN-α production in response to CpG-A. (A) Western blotting analysis of Hsp90 expres- sion in pDCs isolated from HSF-1−/−and HSF-1+/−mice. One rep- resentative blot out of three independent experiments is shown. (B) IFN-α production from pDCs derived from HSF-1+/−or HSF- 1−/−mice was measured using ELISA. Bars show mean + SEM from three independent experiments. **p < 0.01; Student’s t-test. (C) Cell extracts from 293XL-hTLR7-HA and 293XL-hTLR9-HA cells were immunoprecipitated (IP) with anti-HA or anti-Hsp90 and ana- lyzed by immunoblotting (IB) for coprecipitated HA, Hsp70, or Hsp90. HA and Hsp 90 served as loading controls. One repre- sentative blot out of three independent experiments is shown. (D) Purified human pDCs were stimulated for 15 min with CpG-A in the presence (CpG-A + 17-AAG) or absence (CpG-A) of 17-AAG; cell extracts were IP with anti-Hsp90 or anti-TLR9 and analyzed by immunoblotting (IB) for coprecipitated TLR9 and Hsp90. One representative blot out of three independent experiments is shown.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Derivative Assay, Expressing, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 5. Hsp90 is essential for CpG-A-mediated TLR9 translocation to early endosomes and production of IFN-α. Mouse pDCs were trans- fected with siRNA against Hsp90α and Hsp90β twice (days 0 and 2) to knock down both Hsp90α and Hsp90β or control siRNA (Cont.). (A) Western blotting analysis of Hsp90 in Hsp90-knocked-down pDCs. β-actin served as a loading control. One representative blot out of three independent experiments is shown. (B) CpG-A-mediated IFN-α produc- tion by mouse pDCs and Hsp90-knocked-down pDCs was measured by ELISA. Bars show mean + SEM from three independent experiments. (C) Translocation of TLR9 in response to CpG-A administration in pDCs and in Hsp90-knocked-down pDCs using laser confocal microscopy. Original magnification: × 630. (D) Analysis of the localization of TLR9 in pDCs and Hsp90-knocked-down pDCs. Bars show mean + SEM. Data are representative of three independent experiments. **p < 0.01; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Translocation Assay, Knockdown, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 7. Hsp90 multichaperone complex in pDCs in mice that devel- oped SLE. (Input) Expression of Hsp90, p23, Hop in purified pDCs isolated from ICR (control) mouse or MRL/lpr mouse that developed SLE was compared by Western blot analysis. β-Actin served as a loading control. (IP made with 2D12) Status of Hsp90 was analyzed using immunopre- cipitation made with mAb 2D12, which recognizes both complexed and uncomplexed forms of Hsp90, followed by Western blotting detected with antibodies against p23 or Hop. Note that the protein quantity of Hsp90 in pDC isolated from SLE-developed mouse was increased com- pared to that from ICR mouse. One representative blot out of three independent experiments is shown.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Expressing, Isolation, Control, Western Blot

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 8. Serum Hsp90α level is increased in SLE patients and is asso- ciated with disease activity. (A) SLE patients were divided into sub- groups according to SLEDAI scores of 0–5, 6–10, and >10. Hsp90 in sera from 17 patients with SLE, six with mixed connective tissue disease, six with autoimmune vasculitis, ten with Sj¨ogren’s syndrome, ten with Micklickz disease, and six healthy controls was measured using ELISA. Each dot shows an individual patient, bars represent mean. *p < 0.05, **p < 0.01; Student’s t-test. (B) Serum Hsp90α levels before treatment and after treatment were compared in SLE patients (n = 17). ***p < 0.001; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 9. Serum Hsp90 level is dependent on the disease activity of SLE in mouse model. (A) The serum Hs90α level in MRL/lpr mice increased with age in weeks. (B) Comparison of serum Hsp90α level between 17- DMAG-treated mice and PBS-treated mice. Serum Hsp90α levels were determined ELISA. Each dot shows individual mouse, bars represent mean. Statistically significant differences were observed between sub- groups of PBS-treated and 17-DMAG-treated mice. **p < 0.01; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Activity Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

doi: 10.1002/eji.201445293

Figure Lengend Snippet: Figure 10. Extracellular Hsp90 in lupus serum is involved in IFN-α pro- duction by pDCs through binding to self-DNA and anti-DNA Ab. (A) Purified human pDCs (5 × 104/100 μL) were incubated in serum-free RPMI and the serum of an SLE patients (at a final concentration of 50% vol/vol) with or without Hsp90 depletion as shown by Western blotting using anti-Hsp90 antibody. Albumin served as a loading control. One representative blot out of three independent experiments is shown. (B) After 24 h, the concentrations of IFN-α in the culture supernatants were measured by ELISA. (C) Self-DNA or (D) anti-DNA Ab bound to Hsp90 in serum from SLE patients (before treatment and after treatment) was detected using ELISA. (E) Self-DNA purified from active SLE patients serum (10 μg/mL) was mixed with Hsp90 (10 μg/mL) and loaded onto human pDCs for 24 h. IFN-α in culture supernatant was measured. Data show mean + SEM and are representative of three independent exper- iments. *p < 0.01; Student’s t-test.

Article Snippet: A different set of siRNA targeting Hsp90α and Hsp90β (sc 35610) was purchased from Santa Cruz Biotechnology. siRNA was transfected into mouse pDCs twice using Lipofectamine RNAiMAX transfection reagent (Life Technologies) on days 0 and 2.

Techniques: Binding Assay, Incubation, Concentration Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oncology

Article Title: Human bladder cancer cells undergo cisplatin-induced apoptosis that is associated with p53-dependent and p53-independent responses

doi: 10.3892/ijo_00000353

Figure Lengend Snippet: Figure 5. Granzyme B-mediated proteolytic cleavage of Hsp90 after cisplatin administration in human bladder cancer cells: a grade-specific process. As evinced by a Western blotting approach, the higher drug dose was able to significantly induce the Granzyme B protein expression levels and the generation of a major ~65 kDa Hsp90 cleaved fragment (the thick band at the bottom of the bracket) exclusively in RT4 (grade I) but not in T24 (grade III) cisplatin-treated cells.

Article Snippet: The antibodies recognizing Caspase-3 (#9662), Caspase-8 (#9746), Caspase-9 (#9502), PARP (#9542), Lamin A/C (#2032), ßActin (#4968), p53 (#9282), Phospho-p53-Ser15 (#9284), Phospho-p53-Ser392 (#9281), Akt (#9272), Phospho-Akt- Ser473 (#9271), XIAP (#2042) and Granzyme B (#4275) were obtained from Cell Signaling Technology Inc. (Hertfordshire, UK), whereas the antibody against Hsp90 (SC-13119) was purchased from Santa Cruz Biotechnology Inc. (California, USA).

Techniques: Western Blot, Expressing

Journal: Frontiers in Microbiology

Article Title: Identification of a Novel Serum Biomarker for Tuberculosis Infection in Chinese HIV Patients by iTRAQ-Based Quantitative Proteomics

doi: 10.3389/fmicb.2018.00330

Figure Lengend Snippet: Differentially expressed proteins in the sera of HIV positive patients with active tuberculosis (TB) relative to controls (only HIV positive patients).

Article Snippet: The concentrations of ENG (CUSABIO, CSB-E10030h, China), PSMB2 (CUSABIO, CSB-E17836h, China), HSP90AA1 (CUSABIO, CSB-E13462h, China), HSPA8 (CUSABIO, CSB-EL010829HU, China), CHI3L1 (CUSABIO, CSB-E13608h, China), CAP1 (CUSABIO, CSB-EL004486HU, China), ORM1 (CUSABIO, CSB-EL017237HU, China), RETN (CUSABIO, CSB-E06884h, China), LPA (CUSABIO, CSB-EQ028005HU, China) and ZYX (CUSABIO, CSB-EL027165HU, China) in the serum samples were quantified using ELISA according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Identification of a Novel Serum Biomarker for Tuberculosis Infection in Chinese HIV Patients by iTRAQ-Based Quantitative Proteomics

doi: 10.3389/fmicb.2018.00330

Figure Lengend Snippet: Significantly enriched GO terms in the protein–protein interaction network for the differentially expressed proteins.

Article Snippet: The concentrations of ENG (CUSABIO, CSB-E10030h, China), PSMB2 (CUSABIO, CSB-E17836h, China), HSP90AA1 (CUSABIO, CSB-E13462h, China), HSPA8 (CUSABIO, CSB-EL010829HU, China), CHI3L1 (CUSABIO, CSB-E13608h, China), CAP1 (CUSABIO, CSB-EL004486HU, China), ORM1 (CUSABIO, CSB-EL017237HU, China), RETN (CUSABIO, CSB-E06884h, China), LPA (CUSABIO, CSB-EQ028005HU, China) and ZYX (CUSABIO, CSB-EL027165HU, China) in the serum samples were quantified using ELISA according to the manufacturer’s instructions.

Techniques: Cell Surface Receptor Assay

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: 200 pmoles of siRNA specific to HSP90 or the same amounts of control siRNA were transfected to RD cells. After 24 hours of transfection, cells were following infected separately with MOI=0.01 of C2 and B4 genotypes of EV71 5746 and E59 strains. Cells were washed after one hour of infection and cultured for another 16 hours. (A) Cells were lysed and detected the content of HSP90α/β and EV71 capsid proteins by Western blot using anti-HSP90α/β and Mab979 antibodies, respectively. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Corresponding changes in HSP90α/β and EV71 capsid proteins levels compared to the expression levels in the cells with 200 pmoles control siRNA as 1.0 were quantified using Image-Pro Plus 6.0 software. (B) Cells were incubated with anti-HSP90α/β antibody (H-114) or control rabbit IgG (filled curve) at 4°C for another one hour. The cells were washed and fixed by 2% paraformaldehyde in PBS for 30 min. After washing, the treated cells were incubated with anti-rabbit IgG-TR antibody. Stained cells were run on a FACScan flow cytometer and analyzed by using CellQuest software. (C) Cytosolic fractions were prepared as described in the materials and methods and subjected to western blot to detect HSP90α/β as described in (A). Data represent one of two independent experiments. Unpaired student t test with Welch correction was used for statistical analysis (* p <=0.05, ** p <=0.01).

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Control, Transfection, Infection, Cell Culture, Western Blot, Membrane, Expressing, Software, Incubation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: 200 pmoles of HSP90α/β siRNA or the same amounts of control siRNA were transfected to RD cells and cultured for 24 hours, Cells were following infected separately with MOI=0.01 of EV71 5746 and E59 strains. Cells were washed after one hour of infection and cultured for another 12, 24, 36, and 48 hours. After cultivation, the supernatants were harvested and subjected to assay virus titer (TCID 50 ) as described in the Materials and Methods. Data represented one of two independent experiments. Unpaired student t test with Welch correction was calculated.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Control, Transfection, Cell Culture, Infection, Virus

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: RD cells were transfected individually with 200 pmoles of HSP90α, HSP90β, or the control siRNA, and then incubated for 24 hours, Cells were following infected with MOI=0.01 of EV71 5746 strain. Cells were washed after one hour of infection and cultured for another 16 hours. (A) Cells were lysed and detected the EV71 capsid proteins, HSP90α, and HSP90β by Western blot using specific antibodies, respectively. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. (B) The culture mediums were harvested at 12, 24, 36, and 48 hours post infection and applied to detect virus titer (TCID 50 ) as described in the Materials and Methods. Data represented one of two independent experiments. Unpaired student t test with Welch correction was calculated.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Transfection, Control, Incubation, Infection, Cell Culture, Western Blot, Membrane, Virus

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: Pretreatment of RD cells with 2 µM or 0 µM (0.1% DMSO) of GA for one hour before MOI = 0.01 of EV71 5746 (C2) and E59 (B4) infection was conducted individually. After one hour infection, (A) cells were washed with Opti-MEM medium and incubated for 16 hours. Cells lysate were further prepared to detect EV71 capsid protein by western blot using MAB979 antibody. The internal cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. (B) RNA was extracted at 90 min post infection and subjected to quantify EV71 transcripts by real-time RT-PCR using the primers against VP1 region. The relative expression of target gene was calculated as described in the Materials and Methods. Each normalized 2 Ct value from GA-treated cells was ratio to the value from the mean of 2 Ct obtained from the vehicle-treated cells. A schematic representation of the EV71 transcripts expression was shown. Data represented one of two independent experiments. Unpaired student t test with Welch correction was used for statistical analysis. (C) RD cells were treated with various concentration of GA or 0 µm of GA at 37°C for one hour before EV71 (MOI=20) infection for another one hour. After infection, cells were fixed before antibody staining. EV71 was stained with Mab979 antibody. (D) RD cells infected with EV71 (MOI=20) at 4°C for one hour without GA treatment were fixed and stained with Mab979 and anti-HSP90 antibodies (N-17). The respective secondary antibodies conjugated with fluorescence dye described in the Materials and Methods were used. Detection of EV71 (green), HSP90 (red), EV71-HSP90 merging (yellow) and nuclei stained with DAPI (blue) using a confocal microscope Leica TCS SP5 II was performed. The pictures were taken at 200x magnification from 2 independent experiments and one of them was shown.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Infection, Incubation, Western Blot, Membrane, Quantitative RT-PCR, Expressing, Concentration Assay, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: (A) RD cells were treated with 2 µM (gray curve) or 0 µM (0.1% DMSO; black curve) of GA for one hour and then washed with 1xPBS before incubated with anti-HSP90 antibody (N-17) or control goat IgG (filled curve) at 4°C for another one hour. The cells were washed and fixed by 2% paraformaldehyde in PBS for 30 min. After washing, the cells were stained with anti-goat IgG-TR antibody and analyzed by the FACScan flow cytometer. (B) Pretreatment of RD cells with 10 and 20 µg of anti-HSP90 antibody (H-114) or control rabbit IgG (Iso-Ig) at 4°C for one hour before MOI=0.1 of EV71 5746 infection was conducted. After one hour infection, cells were washed with medium and incubated for another 16 hours. Viral capsid proteins in the cells were detected by western blot using MAB979 antibody. The internal β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Corresponding changes in EV71 VP0 levels compared to the expression levels in the cells without antibody treatment as 1.0 were quantified using Image-Pro Plus 6.0 software. (C) Performance of co-immunoprecipitation of EV71 5746 and recombinant HSP90β by anti-EV71 E1 antibody or non-immune mouse IgG (mIgG) following by immunoprecipitation with protein G-agarose was described in the Materials and Methods. Precipitates were washed and subjected to detect the associated EV71 capsid proteins and HSP90 by blotting with MAb979 and anti-HSP90 antibodies, respectively. (D) MOI =0.1 of EV71 5746 was pre-mixed with various amounts of recombinant HSP90β or bovine serum albumin (BSA) and incubated at 4°C for overnight. EV71-HSP90β or EV71-BSA mixtures were inoculated into 12-well plate of RD cells and incubated on ice for 90 min. Followed by washing the unbound EV71 away, total RNA were extracted to quantify EV71 transcripts as described in the legend of Figure 4. Each normalized 2 Ct value from EV71-HSP90β mixtures-treated cells was ratio to the value from the value of 2 Ct obtained from the only EV71-treated cells that represented as 100%. A schematic representation of the percentage of EV71 attachment was shown. Unpaired student t test with Welch correction was used for statistical analysis.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Incubation, Control, Staining, Flow Cytometry, Infection, Western Blot, Membrane, Expressing, Software, Immunoprecipitation, Recombinant

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: RD cells were treated with 2 µM of GA by 30 min early of MOI= 0.01 of EV71 5746 infection and incubated for 16 hours in the presence of drug. Alternatively, cells were treated with GA at 4 or 8 hours post infection and then continued the incubation up to 16 hours. (A) Cell extracts were prepared to detect EV71 capsid or HSP90 proteins by immunoblotting with Mab979 antibody or anti-HSP90 antibody (N-17), respectively. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments. (B) RNA were prepared at 4 and 16 hours post infection to quantify EV71 transcripts as described in the legend of . Each β-actin normalized 2 Ct value from GA-treated cells was ratio to the value from the value of 2 Ct obtained from the vehicle-treated cells. (C) RD cells were treated with vehicle, or 2 µM of GA, or 20 µM of MG132 alone, or GA plus MG132, at 4 hours post EV71 infection and continued the incubation for 12 hours. The extracts were prepared to detect viral capsid proteins by Western blotting using Mab979 antibody. Data represent one of two independent experiments.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Infection, Incubation, Western Blot, Membrane

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: (A) RD cells were treated with vehicle or 2 µM of GA in the presence of MG132 and followed by MOI=0.1 of EV71 5746 infection was performed. Cell lysates were prepared at 16 hours post infection and fractionated in iodixanol density-gradients as described in the Materials and Methods. Each fraction was subjected to Western blot using Mab 979 antibody. (B) RD cells grew on coverslips prior to the infection of EV71 5746 (MOI = 5). After infection, cells were washed with medium and incubated at 37°C for 12 hours before fixed with cold methanol. HSP90 and EV71 were detected by adding of anti-HSP90 (H-114) and MAb979 antibodies and stained with the respective secondary antibodies conjugated with fluorescence dye as described in the Materials and Methods. Detection of cellular HSP90 (red), EV71 (green), and EV71-HSP90 merging (yellow) using a confocal microscope Leica TCS SP5 II was performed. The pictures were taken at 200x or 630x magnification from 2 independent experiments and one of them was shown.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Infection, Western Blot, Incubation, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

doi: 10.1371/journal.pone.0077133

Figure Lengend Snippet: In the infection, EV71 particles attach to the surface receptors (such as human SCARB2 or PSGL-1), HSP90 expressed on the surface may following bind to EV71 and help for virus entry. The newly synthesized viral capsid proteins may bind to HSP90 to avoid being degraded by proteosome that facilitates viral assembling. Targeting of HSP90 using the inhibitor geldanamycin (GA) and its analog, 17-allyamino-17-demethoxygeldanamycin (17-AAG), can efficiently control EV71 infection based on the blockage the described pathways that HSP90 involved.

Article Snippet: Specific siRNA to HSP90α/β gene (Cat. No. 202611C05, Invitrogen, CA, USA) with the control siRNA (Invitrogen, CA, USA), or siRNA specific to HSP90α gene (three oligonucleotides mixture, Cat. No. sc-29353A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) or HSP90β gene (three oligonucleotides mixture, Cat. No. sc-35606A/B/C, Santa Cruz Biotechnology, Inc, CA, USA) with the control siRNA (Santa Cruz Biotechnology, Inc, CA, USA) were used.

Techniques: Infection, Virus, Synthesized, Control