hsc 70 Search Results


hsp72  (Santa Cruz Biotechnology)
95
Santa Cruz Biotechnology hsp72
Fig. 1 | Identification of the co-activator TRRAP that interacts with HSF1. a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty- one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of <t>HSP72</t> mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with
Hsp72, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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h sc 70  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology h sc 70
Fig. 1 | Identification of the co-activator TRRAP that interacts with HSF1. a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty- one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of <t>HSP72</t> mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with
H Sc 70, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hsc70  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology hsc70
FIGURE 5. Hsp70 increases and Hsp90 inhibits nNOS ubiquitination in HEK293Tcells.A,overexpressionofHsp90andHsp70.Cellsweretransfected with cDNAs for His-HA-ubiquitin, CHIP, nNOS, and Hsp90 or Hsp70 or vector plasmid. After 48 h, lysates were prepared and Western blotted for Hsp70, Hsp90, and -tubulin. B, effect of Hsp90 and Hsp70 overexpression on nNOS mono-ubiquitination (mono-Ub). Cells were transfected as above, and lysates were immunoprecipitated with anti-nNOS antibody and Western blotted with anti-nNOS and anti-HA. The upper panel shows the region above mono- ubiquitinated nNOS blotted with anti-HA antibody. The lower panel shows a short exposure for unmodified nNOS protein, and the middle panel shows a long exposure for the mono-ubiquitinated nNOS. The bar graphs show the relative densities of mono-ubiquitinated nNOS bands expressed as means S.E. for three separate experiments. **, p 0.01, *, p 0.05. HA-Ub, HA-ubiquitin. FIGURE 6. Hsp90 increases and Hsp70 decreases level of nNOS protein in HEK293 cells. A, overexpression of Hsp90. HEK293T cells were transfected with cDNAs for CHIP, nNOS, and increasing amounts of Hsp90 as noted above the immunoblot lanes. After 48 h, cell lysates were prepared in SDS sample buffer and aliquots were electrophoresed and Western blotted for nNOS, Hsp90, Hsp70, and -tubulin. B, overexpression of Hsp70. Protocol was as above except for cDNA for Hsp70 instead of Hsp90. C, decreasing expression of Hsp90, <t>Hsc70,</t> and Hsp70. HEK293 cells stably expressing nNOS were trans- fected with scrambled siRNA (lane 1) or siRNA for GAPDH (lane 2), Hsp90/ (lane 3), Hsc70 (lane 4), Hsp70 (lane 5), or both Hsc70 and Hsp70 (lane 6). After 48 h, cell lysates were prepared and Western blotted for nNOS, Hsp90, and GAPDH and with antibodies specific for Hsc70 or Hsp70. The bar graphs show the relative densities of the nNOS bands expressed as the means S.E. for three separate experiments. ***, p 0.001.
Hsc70, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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shrnas against hsc70  (Santa Cruz Biotechnology)
91
Santa Cruz Biotechnology shrnas against hsc70
<t>HSC70</t> directly binds NS5A in vitro through both its nucleotide binding domain (NBD) and substrate binding domain (SBD) and does not bind HSP70. All panels display surface plasmon resonance (SPR) analyses. The immobilized and injected proteins are indicated in figure legends. For all assays, the injection time is 180 seconds followed by 180 seconds of dissociation. In panels A and D–H, the binding signal for human serum albumin (HSA) (negative control) is subtracted from the binding of HSC70 constructs (i.e. HSA binding is set at zero response units). A. Full-length HSC70 directly binds NS5A. B. Human insulin does not bind to NS5A. C. Human insulin does not bind HSP70. D. HSC70-NBD directly binds NS5A. E. HSC70-SBD directly binds NS5A. F. Full-length HSC70 does not bind HSP70. G. HSC70-NBD does not bind HSP70. H. HSC70-SBD does not bind HSP70.
Shrnas Against Hsc70, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hsc 70  (Santa Cruz Biotechnology)
91
Santa Cruz Biotechnology hsc 70
<t>HSC70</t> directly binds NS5A in vitro through both its nucleotide binding domain (NBD) and substrate binding domain (SBD) and does not bind HSP70. All panels display surface plasmon resonance (SPR) analyses. The immobilized and injected proteins are indicated in figure legends. For all assays, the injection time is 180 seconds followed by 180 seconds of dissociation. In panels A and D–H, the binding signal for human serum albumin (HSA) (negative control) is subtracted from the binding of HSC70 constructs (i.e. HSA binding is set at zero response units). A. Full-length HSC70 directly binds NS5A. B. Human insulin does not bind to NS5A. C. Human insulin does not bind HSP70. D. HSC70-NBD directly binds NS5A. E. HSC70-SBD directly binds NS5A. F. Full-length HSC70 does not bind HSP70. G. HSC70-NBD does not bind HSP70. H. HSC70-SBD does not bind HSP70.
Hsc 70, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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heat shock cognate 70 hsc70  (Santa Cruz Biotechnology)
86
Santa Cruz Biotechnology heat shock cognate 70 hsc70
<t>HSC70</t> directly binds NS5A in vitro through both its nucleotide binding domain (NBD) and substrate binding domain (SBD) and does not bind HSP70. All panels display surface plasmon resonance (SPR) analyses. The immobilized and injected proteins are indicated in figure legends. For all assays, the injection time is 180 seconds followed by 180 seconds of dissociation. In panels A and D–H, the binding signal for human serum albumin (HSA) (negative control) is subtracted from the binding of HSC70 constructs (i.e. HSA binding is set at zero response units). A. Full-length HSC70 directly binds NS5A. B. Human insulin does not bind to NS5A. C. Human insulin does not bind HSP70. D. HSC70-NBD directly binds NS5A. E. HSC70-SBD directly binds NS5A. F. Full-length HSC70 does not bind HSP70. G. HSC70-NBD does not bind HSP70. H. HSC70-SBD does not bind HSP70.
Heat Shock Cognate 70 Hsc70, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-hsc-70  (Autogen-Bioclear ltd)
90
Autogen-Bioclear ltd anti-hsc-70
<t>HSC70</t> directly binds NS5A in vitro through both its nucleotide binding domain (NBD) and substrate binding domain (SBD) and does not bind HSP70. All panels display surface plasmon resonance (SPR) analyses. The immobilized and injected proteins are indicated in figure legends. For all assays, the injection time is 180 seconds followed by 180 seconds of dissociation. In panels A and D–H, the binding signal for human serum albumin (HSA) (negative control) is subtracted from the binding of HSC70 constructs (i.e. HSA binding is set at zero response units). A. Full-length HSC70 directly binds NS5A. B. Human insulin does not bind to NS5A. C. Human insulin does not bind HSP70. D. HSC70-NBD directly binds NS5A. E. HSC70-SBD directly binds NS5A. F. Full-length HSC70 does not bind HSP70. G. HSC70-NBD does not bind HSP70. H. HSC70-SBD does not bind HSP70.
Anti Hsc 70, supplied by Autogen-Bioclear ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hsc 70 crispr activation plasmid  (Santa Cruz Biotechnology)
N/A
CRISPR/Cas9 KO Plasmids consists of HSC 70-specific 20 nt guide RNA sequences derived from the GeCKO (v2) library. For CRISPR gene knockout, gRNA sequences direct the Cas9 protein to induce a site-specific double strand break
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hsc 70 crispr/cas9 ko plasmid  (Santa Cruz Biotechnology)
N/A
CRISPR/Cas9 KO Plasmids consists of HSC 70-specific 20 nt guide RNA sequences derived from the GeCKO (v2) library. For CRISPR gene knockout, gRNA sequences direct the Cas9 protein to induce a site-specific double strand break
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hsc 70 (m)-pr  (Santa Cruz Biotechnology)
N/A
Gene Silencers generally consist of pools of three to five target-specific 19-25 nucleotide sequences in length. For independent verification of HSC 70 gene silencing results, individual duplex components or plasmids are also available upon request.
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hsc 70 double nickase plasmid  (Santa Cruz Biotechnology)
N/A
CRISPR/Cas9 KO Plasmids consists of HSC 70-specific 20 nt guide RNA sequences derived from the GeCKO (v2) library. For CRISPR gene knockout, gRNA sequences direct the Cas9 protein to induce a site-specific double strand break
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Image Search Results


Fig. 1 | Identification of the co-activator TRRAP that interacts with HSF1. a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty- one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of HSP72 mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with

Journal: Nature communications

Article Title: HSF1 phosphorylation establishes an active chromatin state via the TRRAP-TIP60 complex and promotes tumorigenesis.

doi: 10.1038/s41467-022-32034-4

Figure Lengend Snippet: Fig. 1 | Identification of the co-activator TRRAP that interacts with HSF1. a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty- one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of HSP72 mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with

Article Snippet: The following antibodies were used: rabbit antibodies for mHSF1 (antimHSF1n, Nakai lab, 1/1000), hTRRAP (anti-hTRRAP-2, Nakai lab, 1/ 1000), hTRIM24 (anti-hTRIM24-3, Nakai lab, 1/1000), hTRIM33 (antihTRIM33-2, Nakai lab, 1/1000), hPLK1 (anti-hPLK1-1, Nakai lab, 1/1000), GST (anti-GST, Nakai lab, 1/1000),mHSP110 (anti-mHSP110a, Nakai lab, 1/1000), hHSP90 (anti-hHSF90d, Nakai lab, 1/1000), hHSP40 (antihHSP40a,Nakai lab, 1/1000)15, p300 (SantaCruz, sc-585, 1/1000), p400 (Novus Biologicals, NB200-210, 1/1000), H2B (Abcam, ab1790, 1/ 1000), ubiquityl-histone H2B (Lys120) (Cell Signaling, 5546, 1/1000), RNF40 (GeneTex, GTX115295, 1/1000), histone H3 (Abcam, ab1791, 1/ 1000), H3 (acetyl K18) (Abcam, ab1191, 1/1000), Lamin B1 (Abcam, Ab16048, 1/1000), HSF1 (phosphor S326) (Abcam, ab115702, 1/1000) and HSF1 (phosphoS419) (anti-HSF1 phospho-S419b, Nakai lab, 1/ 1000), mouse antibodies for PLK1 (Santa Cruz, sc-17783, 1/1000), HSP72 (Santa Cruz, sc-24, 1/1000), ubiquitin (Santa Cruz, sc-8017, 1/ 1000), CSNK1A1 (Santa Cruz, sc-74582, 1/1000), NEK7 (Santa Cruz, sc393539, 1/1000), RNF20 (Santa Cruz, sc-517358, 1/1000), β-actin (Milipore Sigma, A5441, 1/1000), and GFP (Nacalai Tesque, GF200, 1/ 1000), rat antibody for HA (Roche, ROAHAHA, 1/1000), and goat Nature Communications | (2022) 13:4355 12 antibody for TIP60 (Santa Cruz, N-17, 1/200).

Techniques: Immunoprecipitation, Western Blot, Expressing, Control, ChIP-sequencing, Binding Assay

FIGURE 5. Hsp70 increases and Hsp90 inhibits nNOS ubiquitination in HEK293Tcells.A,overexpressionofHsp90andHsp70.Cellsweretransfected with cDNAs for His-HA-ubiquitin, CHIP, nNOS, and Hsp90 or Hsp70 or vector plasmid. After 48 h, lysates were prepared and Western blotted for Hsp70, Hsp90, and -tubulin. B, effect of Hsp90 and Hsp70 overexpression on nNOS mono-ubiquitination (mono-Ub). Cells were transfected as above, and lysates were immunoprecipitated with anti-nNOS antibody and Western blotted with anti-nNOS and anti-HA. The upper panel shows the region above mono- ubiquitinated nNOS blotted with anti-HA antibody. The lower panel shows a short exposure for unmodified nNOS protein, and the middle panel shows a long exposure for the mono-ubiquitinated nNOS. The bar graphs show the relative densities of mono-ubiquitinated nNOS bands expressed as means S.E. for three separate experiments. **, p 0.01, *, p 0.05. HA-Ub, HA-ubiquitin. FIGURE 6. Hsp90 increases and Hsp70 decreases level of nNOS protein in HEK293 cells. A, overexpression of Hsp90. HEK293T cells were transfected with cDNAs for CHIP, nNOS, and increasing amounts of Hsp90 as noted above the immunoblot lanes. After 48 h, cell lysates were prepared in SDS sample buffer and aliquots were electrophoresed and Western blotted for nNOS, Hsp90, Hsp70, and -tubulin. B, overexpression of Hsp70. Protocol was as above except for cDNA for Hsp70 instead of Hsp90. C, decreasing expression of Hsp90, Hsc70, and Hsp70. HEK293 cells stably expressing nNOS were trans- fected with scrambled siRNA (lane 1) or siRNA for GAPDH (lane 2), Hsp90/ (lane 3), Hsc70 (lane 4), Hsp70 (lane 5), or both Hsc70 and Hsp70 (lane 6). After 48 h, cell lysates were prepared and Western blotted for nNOS, Hsp90, and GAPDH and with antibodies specific for Hsc70 or Hsp70. The bar graphs show the relative densities of the nNOS bands expressed as the means S.E. for three separate experiments. ***, p 0.001.

Journal: Journal of Biological Chemistry

Article Title: Modulation of Heme/Substrate Binding Cleft of Neuronal Nitric-oxide Synthase (nNOS) Regulates Binding of Hsp90 and Hsp70 Proteins and nNOS Ubiquitination

doi: 10.1074/jbc.m111.323295

Figure Lengend Snippet: FIGURE 5. Hsp70 increases and Hsp90 inhibits nNOS ubiquitination in HEK293Tcells.A,overexpressionofHsp90andHsp70.Cellsweretransfected with cDNAs for His-HA-ubiquitin, CHIP, nNOS, and Hsp90 or Hsp70 or vector plasmid. After 48 h, lysates were prepared and Western blotted for Hsp70, Hsp90, and -tubulin. B, effect of Hsp90 and Hsp70 overexpression on nNOS mono-ubiquitination (mono-Ub). Cells were transfected as above, and lysates were immunoprecipitated with anti-nNOS antibody and Western blotted with anti-nNOS and anti-HA. The upper panel shows the region above mono- ubiquitinated nNOS blotted with anti-HA antibody. The lower panel shows a short exposure for unmodified nNOS protein, and the middle panel shows a long exposure for the mono-ubiquitinated nNOS. The bar graphs show the relative densities of mono-ubiquitinated nNOS bands expressed as means S.E. for three separate experiments. **, p 0.01, *, p 0.05. HA-Ub, HA-ubiquitin. FIGURE 6. Hsp90 increases and Hsp70 decreases level of nNOS protein in HEK293 cells. A, overexpression of Hsp90. HEK293T cells were transfected with cDNAs for CHIP, nNOS, and increasing amounts of Hsp90 as noted above the immunoblot lanes. After 48 h, cell lysates were prepared in SDS sample buffer and aliquots were electrophoresed and Western blotted for nNOS, Hsp90, Hsp70, and -tubulin. B, overexpression of Hsp70. Protocol was as above except for cDNA for Hsp70 instead of Hsp90. C, decreasing expression of Hsp90, Hsc70, and Hsp70. HEK293 cells stably expressing nNOS were trans- fected with scrambled siRNA (lane 1) or siRNA for GAPDH (lane 2), Hsp90/ (lane 3), Hsc70 (lane 4), Hsp70 (lane 5), or both Hsc70 and Hsp70 (lane 6). After 48 h, cell lysates were prepared and Western blotted for nNOS, Hsp90, and GAPDH and with antibodies specific for Hsc70 or Hsp70. The bar graphs show the relative densities of the nNOS bands expressed as the means S.E. for three separate experiments. ***, p 0.001.

Article Snippet: Small interfering RNAs (siRNAs) for Hsp90 / , Hsc70, and Hsp70, the K19 rabbit polyclonal antibody specific for Hsc70, and anti-HA antibodyconjugated agarose were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Ubiquitin Proteomics, Plasmid Preparation, Western Blot, Over Expression, Transfection, Immunoprecipitation, Expressing, Stable Transfection

HSC70 directly binds NS5A in vitro through both its nucleotide binding domain (NBD) and substrate binding domain (SBD) and does not bind HSP70. All panels display surface plasmon resonance (SPR) analyses. The immobilized and injected proteins are indicated in figure legends. For all assays, the injection time is 180 seconds followed by 180 seconds of dissociation. In panels A and D–H, the binding signal for human serum albumin (HSA) (negative control) is subtracted from the binding of HSC70 constructs (i.e. HSA binding is set at zero response units). A. Full-length HSC70 directly binds NS5A. B. Human insulin does not bind to NS5A. C. Human insulin does not bind HSP70. D. HSC70-NBD directly binds NS5A. E. HSC70-SBD directly binds NS5A. F. Full-length HSC70 does not bind HSP70. G. HSC70-NBD does not bind HSP70. H. HSC70-SBD does not bind HSP70.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: HSC70 directly binds NS5A in vitro through both its nucleotide binding domain (NBD) and substrate binding domain (SBD) and does not bind HSP70. All panels display surface plasmon resonance (SPR) analyses. The immobilized and injected proteins are indicated in figure legends. For all assays, the injection time is 180 seconds followed by 180 seconds of dissociation. In panels A and D–H, the binding signal for human serum albumin (HSA) (negative control) is subtracted from the binding of HSC70 constructs (i.e. HSA binding is set at zero response units). A. Full-length HSC70 directly binds NS5A. B. Human insulin does not bind to NS5A. C. Human insulin does not bind HSP70. D. HSC70-NBD directly binds NS5A. E. HSC70-SBD directly binds NS5A. F. Full-length HSC70 does not bind HSP70. G. HSC70-NBD does not bind HSP70. H. HSC70-SBD does not bind HSP70.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: In Vitro, Binding Assay, SPR Assay, Injection, Negative Control, Construct

HSC70 interacts with NS5A in vivo. Coimmunoprecipitation assay demonstrates that HSC70 binds NS5A in infected cells. Huh-7.5 cells were infected for 72 hours and harvested. Cell lysates from infected and uninfected control cells were immunoprecipitated with antibody against HSC70 followed by Western analysis.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: HSC70 interacts with NS5A in vivo. Coimmunoprecipitation assay demonstrates that HSC70 binds NS5A in infected cells. Huh-7.5 cells were infected for 72 hours and harvested. Cell lysates from infected and uninfected control cells were immunoprecipitated with antibody against HSC70 followed by Western analysis.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: In Vivo, Co-Immunoprecipitation Assay, Infection, Control, Immunoprecipitation, Western Blot

HSC70 colocalizes with NS5A in vivo. Immunofluorescence assay demonstrates colocalization of HSC70 and NS5A in vivo. Huh-7.5 cells were infected for 48 hours followed by simultaneous immunofluorescence staining with antibodies against HSC70 and NS5A. A. HSC70 (blue). B. NS5A (red). C. Immunofluorescence staining for both HSC70 and NS5A (pink). D. Scatter plot demonstrating the colocalization of HSC70 and NS5A. E. Table indicating Manders overlap coefficients for colocalization of HSC70 with NS5A (M1) and colocalization of NS5A with HSC70 (M2). Overlap analyses were done on four independent immunofluorescence experiments.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: HSC70 colocalizes with NS5A in vivo. Immunofluorescence assay demonstrates colocalization of HSC70 and NS5A in vivo. Huh-7.5 cells were infected for 48 hours followed by simultaneous immunofluorescence staining with antibodies against HSC70 and NS5A. A. HSC70 (blue). B. NS5A (red). C. Immunofluorescence staining for both HSC70 and NS5A (pink). D. Scatter plot demonstrating the colocalization of HSC70 and NS5A. E. Table indicating Manders overlap coefficients for colocalization of HSC70 with NS5A (M1) and colocalization of NS5A with HSC70 (M2). Overlap analyses were done on four independent immunofluorescence experiments.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: In Vivo, Immunofluorescence, Infection, Staining

siRNA-mediated knockdown of HSC70 is not cytotoxic to cells. A. MTT assay demonstrates that knockdown of HSC70 does not have any cytotoxic effects at 96 hours after siRNA transfection. B. Western analysis of cells treated with the above siRNAs. C. Densitometry of the Western blot in panel B.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: siRNA-mediated knockdown of HSC70 is not cytotoxic to cells. A. MTT assay demonstrates that knockdown of HSC70 does not have any cytotoxic effects at 96 hours after siRNA transfection. B. Western analysis of cells treated with the above siRNAs. C. Densitometry of the Western blot in panel B.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: Knockdown, MTT Assay, Transfection, Western Blot

siRNA-mediated knockdown of HSC70 significantly decreases virus production. A. Intracellular virus production assay demonstrates that knockdown of HSC70 results in a significant decrease in intracellular virus levels. B. Long term infectious virion secretion assay indicates that knockdown of HSC70 dramatically reduced infectious virion secretion.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: siRNA-mediated knockdown of HSC70 significantly decreases virus production. A. Intracellular virus production assay demonstrates that knockdown of HSC70 results in a significant decrease in intracellular virus levels. B. Long term infectious virion secretion assay indicates that knockdown of HSC70 dramatically reduced infectious virion secretion.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: Knockdown, Virus

shRNA-mediated knockdown of HSC70 significantly decreases virus production. A. Western analysis of cells treated with shRNAs against HSC70, HSP70, or both. B. Densitometry of the Western blot in panel A. C. Intracellular virus production assay demonstrates that knockdown of HSC70 results in a significant decrease in intracellular viral levels.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: shRNA-mediated knockdown of HSC70 significantly decreases virus production. A. Western analysis of cells treated with shRNAs against HSC70, HSP70, or both. B. Densitometry of the Western blot in panel A. C. Intracellular virus production assay demonstrates that knockdown of HSC70 results in a significant decrease in intracellular viral levels.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: shRNA, Knockdown, Virus, Western Blot

Knockdown of HSC70 does not affect viral RNA replication and NS5A-augmented IRES-mediated translation. A. Quantitative reverse transcriptase PCR (qRT-PCR) analysis indicates that knockdown of HSC70 does not affect viral RNA replication. B. Schematic of the bicistronic reporter construct used in the dual luciferase assays in part C. This construct contains the Renilla luciferase and Firefly luciferase ORFs under the control of a 5′-cap and the HCV IRES, respectively. The ratio of Firefly to Renilla measurements is indicative of IRES-mediated translation. C. IRES assay indicates that knockdown of HSC70 does not decrease NS5A-augmented IRES-mediated translation.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: Knockdown of HSC70 does not affect viral RNA replication and NS5A-augmented IRES-mediated translation. A. Quantitative reverse transcriptase PCR (qRT-PCR) analysis indicates that knockdown of HSC70 does not affect viral RNA replication. B. Schematic of the bicistronic reporter construct used in the dual luciferase assays in part C. This construct contains the Renilla luciferase and Firefly luciferase ORFs under the control of a 5′-cap and the HCV IRES, respectively. The ratio of Firefly to Renilla measurements is indicative of IRES-mediated translation. C. IRES assay indicates that knockdown of HSC70 does not decrease NS5A-augmented IRES-mediated translation.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: Knockdown, Reverse Transcription, Quantitative RT-PCR, Construct, Luciferase, Control

Knockdown of HSC70 does not affect intracellular viral protein production. A. Intracellular viral protein production assay indicates that knockdown of HSC70 does not decrease intracellular viral protein production. Huh-7.5 cells were treated with siRNAs against HSC70, HSP70, or their corresponding control siRNAs and infected with the reporter virus 24 hours later. At the indicated time points after infection, cells were harvested and luciferase activity was measured. B. Bar graph corresponding to the 24 hours time point of the above assay.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: Knockdown of HSC70 does not affect intracellular viral protein production. A. Intracellular viral protein production assay indicates that knockdown of HSC70 does not decrease intracellular viral protein production. Huh-7.5 cells were treated with siRNAs against HSC70, HSP70, or their corresponding control siRNAs and infected with the reporter virus 24 hours later. At the indicated time points after infection, cells were harvested and luciferase activity was measured. B. Bar graph corresponding to the 24 hours time point of the above assay.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: Knockdown, Control, Infection, Virus, Luciferase, Activity Assay

Knockdown of HSC70 significantly reduces intracellular infectious virion assembly. A. Intracellular infectious virion assembly assay demonstrates that knockdown of HSC70 significantly inhibits virion assembly. B. Short term infectious virion secretion assay demonstrates the effect of knockdown of HSC70 on virus secretion.

Journal: Virology

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle

doi: 10.1016/j.virol.2014.02.016

Figure Lengend Snippet: Knockdown of HSC70 significantly reduces intracellular infectious virion assembly. A. Intracellular infectious virion assembly assay demonstrates that knockdown of HSC70 significantly inhibits virion assembly. B. Short term infectious virion secretion assay demonstrates the effect of knockdown of HSC70 on virus secretion.

Article Snippet: siRNAs against HSC70 (Santa Cruz Biotech, sc-29349) and HSP70 (Thermo Scientific, M-005168-01-0005) as well as Control siRNA-A (Santa Cruz Biotech, sc-37007) and siGENOME Non-Targeting siRNA Pool #2 (Thermo Scientific, D-001206-14-05) were transfected into huh-7.5 cells using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) according to manufacturer’s instructions. shRNAs against HSC70 (Santa Cruz Biotech, sc-29349-SH) and HSP70 (Santa Cruz Biotech, sc-29352-SH) as well as control shRNA plasmid-A (Santa Cruz Biotech, sc-108060) were transfected into huh-7.5 cells using X-tremeGene 9 DNA Transfection Reagent (Roche, 6365779001) according to manufacturer’s instructions.

Techniques: Knockdown, Virus