hsp 70 Stressgen Inc Search Results


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    Thermo Fisher alexa fluor 488
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    Cell Signaling Technology Inc akt
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    Cell Signaling Technology Inc hsp70
    Molecular chaperones facilitate p53 R175H-TAp73α complex formation ( A ) Double immunoprecipitation experiments (Two step Co-IP). H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and <t>HA-HSP70.</t> After 24 h cellular proteins were cross-linked and the first co-immunoprecipitation (1) with anti-p53 antibody was carried out. Protein complexes were eluted from beads (Cell lysates 2) and second co-immunoprecipitation (2) with anti-HA antibody was performed. Laemmli buffer was supplemented with DTT to reverse cross-linking reaction. Lysates from cells transfected with plasmids encoding TAp73α and HA-HSP70 but not p53 R175H were used as a control of the specificity of anti-p53 antibody (first lane, right panel). ( B ) H1299 cells were transfected with plasmids encoding p53 R175H and TAp73α (left panel) or p53 R175H, TAp73α and HA-HSP70 (right panel). Immunoprecipitations were carried out with anti-p53 (left panel) or anti-HA antibody (right panel) to immunoprecipitate p53 or HA-HSP70, respectively. As a control of the specificity of antibodies applied for immunoprecipitations, cell lysates with no p53 R175H or no HA-HSP70 were used (–). ( C ) H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and HA-HSP70 WT/K71S/D10S respectively. 24 h post-transfection cells were lysed and p53 protein was immunoprecipitated with anti-p53 antibody – top panel, or with anti-HA antibody – bottom panel. The immunoprecipitated protein complexes were analyzed by Western blot. ( D ) H1299-R175H cells were transfected with a plasmid encoding HA-HSP70 D10S or with a control plasmid (pcDNA). After 6 h the medium was supplemented with 0.5 μM Ponasterone A (Pon A) to induce p53 R175H. Cells with no induction were treated with DMSO. After 24 h treatment with 60 μM Cisplatin, the apoptotic response of cells stained with Annexin V/Gel Green dye was measured by FACS. Bars represent the decrease (%) of cells in early apoptosis (Annexin V positive, Gel Green dye negative), normalized to non-treated control. Statistical significance ( P value) was counted for three independent experiments with Anova statistical test. ** indicates statistical significance p
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    Cell Signaling Technology Inc anti hsp70
    HSP27 and <t>HSP70</t> expression (WB) at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10) (3 wk study) Values are means ± SD; BFRE: n = 10 at Mid8 n = 9; LLE: n = 7.
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    Cell Signaling Technology Inc cleaved caspase 3
    Activation state of <t>caspase-3</t> in response to nitric oxide. INS832/13 cells were treated for 1 h with 1 m m DEANO or with DEANO followed by washing to remove the donor and an additional 5 h or for 5 h with camptothecin as a positive control for apoptosis.
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    Cell Signaling Technology Inc caspase 3
    Activation state of <t>caspase-3</t> in response to nitric oxide. INS832/13 cells were treated for 1 h with 1 m m DEANO or with DEANO followed by washing to remove the donor and an additional 5 h or for 5 h with camptothecin as a positive control for apoptosis.
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    Cell Signaling Technology Inc rabbit
    Activation state of <t>caspase-3</t> in response to nitric oxide. INS832/13 cells were treated for 1 h with 1 m m DEANO or with DEANO followed by washing to remove the donor and an additional 5 h or for 5 h with camptothecin as a positive control for apoptosis.
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    Stressgen Biotechnologies hsp70
    Double immunostaining confocal microscopy of <t>HSP70</t> and Iba-1 or GFAP in P7 brain sections after HI. Brain sections were from P7 rats subjected to HI followed by 4 h of recovery (P7 4hR). Upper: HSP70 immunostaining (left, green) and microglia marker Iba-1 immunostaining (middle, red) are overlapped each other (right). Arrows point to immunopositive microglia. Lower: HSP70 immunostaining (left, green) and astrocyte marker GFAP immunostaining (middle, red) are not overlapped each other (right). Arrows point to immunopositive microglia. Arrowheads indicate astrocytes. Scale bar = 20 μm.
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    Cell Signaling Technology Inc anti hsp90
    Protein levels of <t>Hsp90,</t> Hsp70, and Hsp27. Control and/or heat shocked utricles from CBA mice, Hsp70 -overexpressing mice, and wild-type littermates were collected 6 h after heat shock. Protein lysates were subjected to SDS-PAGE and then transferred
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    Cell Signaling Technology Inc parp
    Protein levels of <t>Hsp90,</t> Hsp70, and Hsp27. Control and/or heat shocked utricles from CBA mice, Hsp70 -overexpressing mice, and wild-type littermates were collected 6 h after heat shock. Protein lysates were subjected to SDS-PAGE and then transferred
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    Cell Signaling Technology Inc phospho akt
    EGCG modifies the interaction of <t>hsp90</t> with cochaperones, and leads to the degradation of the hsp90 client proteins ErbB2, Raf-1 and <t>phospho-AKT.</t> A. Hepa cytosol was incubated with DMSO or 200μM EGCG (E) for 1h at RT and immunoprecipitated with
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    Cell Signaling Technology Inc anti caspase 3
    EGCG modifies the interaction of <t>hsp90</t> with cochaperones, and leads to the degradation of the hsp90 client proteins ErbB2, Raf-1 and <t>phospho-AKT.</t> A. Hepa cytosol was incubated with DMSO or 200μM EGCG (E) for 1h at RT and immunoprecipitated with
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    Stressgen Biotechnologies anti hsp70
    Effects of S100 proteins on the CHIP-client protein interactions in vitro . A–E , upper panel , GST-CHIP (20 μg), S100 proteins (20 μg each) and appropriate client proteins were mixed with glutathione-Sepharose 4B in the presence of CaCl 2 or EGTA. Control assay ( CHIP− ) was conducted using GST (20 μg). S100 proteins used were as follows: S100A2, S100A6, S100P, S100A12, and control (−). The client proteins were as follows: purified 20 μg of <t>Hsp70</t> ( A ), 40 μg of Hsp90 ( B ), 20 μg of Smad1 ( C ), 20 μg of HSF1 ( D ), and 20 μg of UbcH5a ( E ). Details of the GST pulldown assay are described under “Experimental Procedures.” The resulting samples were subjected to SDS-PAGE and visualized by Western blotting ( WB) using the indicated antibodies. Lower panel , the binding levels of these client proteins to CHIP without S100 proteins ( lane 1 ) were designated as 100% ( Control ), and the relative binding levels ( % of Control ) were plotted. The error bars represent the S.E. with n = 3.
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    Cell Signaling Technology Inc anti hsp27
    Effects of S100 proteins on the CHIP-client protein interactions in vitro . A–E , upper panel , GST-CHIP (20 μg), S100 proteins (20 μg each) and appropriate client proteins were mixed with glutathione-Sepharose 4B in the presence of CaCl 2 or EGTA. Control assay ( CHIP− ) was conducted using GST (20 μg). S100 proteins used were as follows: S100A2, S100A6, S100P, S100A12, and control (−). The client proteins were as follows: purified 20 μg of <t>Hsp70</t> ( A ), 40 μg of Hsp90 ( B ), 20 μg of Smad1 ( C ), 20 μg of HSF1 ( D ), and 20 μg of UbcH5a ( E ). Details of the GST pulldown assay are described under “Experimental Procedures.” The resulting samples were subjected to SDS-PAGE and visualized by Western blotting ( WB) using the indicated antibodies. Lower panel , the binding levels of these client proteins to CHIP without S100 proteins ( lane 1 ) were designated as 100% ( Control ), and the relative binding levels ( % of Control ) were plotted. The error bars represent the S.E. with n = 3.
    Anti Hsp27, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc erk1 2
    FN-Integrin signaling inhibitor G RGD SP and <t>ERK1/2</t> kinase inhibitor PD98059 attenuate GLN-mediated activation of HSF-1. A) IEC-6 cells were treated with 0 mM or 10 mM GLN with or without 1 h prior PD98059 or G RGD SP treatment. Representative Western blot from 3 independent experiments shows [S(P) 303 ]HSF-1 and total HSF-1 levels. B) Densitometric analysis of HSF-1 phosphorylation at serine 303 was measured as mean fold changes relative to total HSF-1±SEM (n = 3). C) Total HSF-1 levels were measured after 3 h recovery after non-lethal HS. Results are displayed as fold change±SEM ratioed to HS 0 mM GLN groups (n = 3).
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    Assay Designs Inc hsp70
    Endoscopic and histological grading of DSS colitis in WT and <t>Hsp70</t> TG mice. WT and Hsp70 UTR-less TG mice were given 3% DSS for 7 days and then given tap water for another 3 days before analysis. A : colonoscopy was performed before and after DSS
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    Santa Cruz Biotechnology hsp70
    Identification of miRNAs involved in gefitinib-dependent suppression of expression of <t>HSP70.</t> A549 cells were incubated with the indicated concentration of gefitinib for 3 h (A). A549 cells were transfected with siRNA for Dicer1 (siDicer1) or non-specific siRNA (ns) and after 24 h were incubated with the indicated concentration of gefitinib for 3 h (B). A549 cells were transfected with the indicated amount ( µg/well) of miRNA mimic RNA fragments for 24 h (C, E). The RNA (A, B, E) and protein (C) expression was monitored and expressed as described in the legend of Fig. 1 . The RUN44 non-coding RNA was used for normalization (A, B, E). The intensities of the HSP70 bands were determined and are expressed relative to the control (one of the gels is shown in C) (D). Values shown are mean ± S.D. ( n = 3). ** P
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    Santa Cruz Biotechnology anti actin
    Identification of miRNAs involved in gefitinib-dependent suppression of expression of <t>HSP70.</t> A549 cells were incubated with the indicated concentration of gefitinib for 3 h (A). A549 cells were transfected with siRNA for Dicer1 (siDicer1) or non-specific siRNA (ns) and after 24 h were incubated with the indicated concentration of gefitinib for 3 h (B). A549 cells were transfected with the indicated amount ( µg/well) of miRNA mimic RNA fragments for 24 h (C, E). The RNA (A, B, E) and protein (C) expression was monitored and expressed as described in the legend of Fig. 1 . The RUN44 non-coding RNA was used for normalization (A, B, E). The intensities of the HSP70 bands were determined and are expressed relative to the control (one of the gels is shown in C) (D). Values shown are mean ± S.D. ( n = 3). ** P
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    Cell Signaling Technology Inc anti caspase 9
    Hsp90β (S226A/S255A) inhibits Apaf-1 oligomerization and <t>caspase-9</t> recruitment. Recombinant Apaf-1 (0.4 μM) was mixed with catalytically inactive caspase-9 [C9 (C287A)] (0.8 μM) and recombinant Hsp90β (1 μM; S226E/S255E
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    Stressgen Biotechnologies hsp90
    Hsp90β (S226A/S255A) inhibits Apaf-1 oligomerization and <t>caspase-9</t> recruitment. Recombinant Apaf-1 (0.4 μM) was mixed with catalytically inactive caspase-9 [C9 (C287A)] (0.8 μM) and recombinant Hsp90β (1 μM; S226E/S255E
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    Cell Signaling Technology Inc 4ebp1
    Effect of HSP90 inhibition on downstream signaling. BON1, NCI-H727 and GOT1 cells were treated with increasing concentrations (1–100 nM) of the HSP90 inhibitor AUY922 (left panel) or HSP990 (right panel) for 24 h. Subsequently, the expression of pErk1/2, Erk1/2, pAkt, Akt, pp70S6K, p70S6K, p4EBP1 and <t>4EBP1</t> was evaluated by western blot analysis. A representative blot out of 3 independently performed experiments is shown.
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    Thermo Fisher bicinchoninic acid method
    Effect of HSP90 inhibition on downstream signaling. BON1, NCI-H727 and GOT1 cells were treated with increasing concentrations (1–100 nM) of the HSP90 inhibitor AUY922 (left panel) or HSP990 (right panel) for 24 h. Subsequently, the expression of pErk1/2, Erk1/2, pAkt, Akt, pp70S6K, p70S6K, p4EBP1 and <t>4EBP1</t> was evaluated by western blot analysis. A representative blot out of 3 independently performed experiments is shown.
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    Cell Signaling Technology Inc anti erk1 2
    A, Odorant-induced <t>Erk1/2</t> activation. Cultures were treated with vehicle, forskolin (FSK), 8-br-cAMP or odorants in the presence or absence of U0126. Odorants increased pErk1/2 levels, whereas forskolin and 8-br-cAMP did not. Preincubation with U0126
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    Image Search Results


    Molecular chaperones facilitate p53 R175H-TAp73α complex formation ( A ) Double immunoprecipitation experiments (Two step Co-IP). H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and HA-HSP70. After 24 h cellular proteins were cross-linked and the first co-immunoprecipitation (1) with anti-p53 antibody was carried out. Protein complexes were eluted from beads (Cell lysates 2) and second co-immunoprecipitation (2) with anti-HA antibody was performed. Laemmli buffer was supplemented with DTT to reverse cross-linking reaction. Lysates from cells transfected with plasmids encoding TAp73α and HA-HSP70 but not p53 R175H were used as a control of the specificity of anti-p53 antibody (first lane, right panel). ( B ) H1299 cells were transfected with plasmids encoding p53 R175H and TAp73α (left panel) or p53 R175H, TAp73α and HA-HSP70 (right panel). Immunoprecipitations were carried out with anti-p53 (left panel) or anti-HA antibody (right panel) to immunoprecipitate p53 or HA-HSP70, respectively. As a control of the specificity of antibodies applied for immunoprecipitations, cell lysates with no p53 R175H or no HA-HSP70 were used (–). ( C ) H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and HA-HSP70 WT/K71S/D10S respectively. 24 h post-transfection cells were lysed and p53 protein was immunoprecipitated with anti-p53 antibody – top panel, or with anti-HA antibody – bottom panel. The immunoprecipitated protein complexes were analyzed by Western blot. ( D ) H1299-R175H cells were transfected with a plasmid encoding HA-HSP70 D10S or with a control plasmid (pcDNA). After 6 h the medium was supplemented with 0.5 μM Ponasterone A (Pon A) to induce p53 R175H. Cells with no induction were treated with DMSO. After 24 h treatment with 60 μM Cisplatin, the apoptotic response of cells stained with Annexin V/Gel Green dye was measured by FACS. Bars represent the decrease (%) of cells in early apoptosis (Annexin V positive, Gel Green dye negative), normalized to non-treated control. Statistical significance ( P value) was counted for three independent experiments with Anova statistical test. ** indicates statistical significance p

    Journal: Oncotarget

    Article Title: Molecular chaperones in the acquisition of cancer cell chemoresistance with mutated TP53 and MDM2 up-regulation

    doi: 10.18632/oncotarget.18899

    Figure Lengend Snippet: Molecular chaperones facilitate p53 R175H-TAp73α complex formation ( A ) Double immunoprecipitation experiments (Two step Co-IP). H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and HA-HSP70. After 24 h cellular proteins were cross-linked and the first co-immunoprecipitation (1) with anti-p53 antibody was carried out. Protein complexes were eluted from beads (Cell lysates 2) and second co-immunoprecipitation (2) with anti-HA antibody was performed. Laemmli buffer was supplemented with DTT to reverse cross-linking reaction. Lysates from cells transfected with plasmids encoding TAp73α and HA-HSP70 but not p53 R175H were used as a control of the specificity of anti-p53 antibody (first lane, right panel). ( B ) H1299 cells were transfected with plasmids encoding p53 R175H and TAp73α (left panel) or p53 R175H, TAp73α and HA-HSP70 (right panel). Immunoprecipitations were carried out with anti-p53 (left panel) or anti-HA antibody (right panel) to immunoprecipitate p53 or HA-HSP70, respectively. As a control of the specificity of antibodies applied for immunoprecipitations, cell lysates with no p53 R175H or no HA-HSP70 were used (–). ( C ) H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and HA-HSP70 WT/K71S/D10S respectively. 24 h post-transfection cells were lysed and p53 protein was immunoprecipitated with anti-p53 antibody – top panel, or with anti-HA antibody – bottom panel. The immunoprecipitated protein complexes were analyzed by Western blot. ( D ) H1299-R175H cells were transfected with a plasmid encoding HA-HSP70 D10S or with a control plasmid (pcDNA). After 6 h the medium was supplemented with 0.5 μM Ponasterone A (Pon A) to induce p53 R175H. Cells with no induction were treated with DMSO. After 24 h treatment with 60 μM Cisplatin, the apoptotic response of cells stained with Annexin V/Gel Green dye was measured by FACS. Bars represent the decrease (%) of cells in early apoptosis (Annexin V positive, Gel Green dye negative), normalized to non-treated control. Statistical significance ( P value) was counted for three independent experiments with Anova statistical test. ** indicates statistical significance p

    Article Snippet: Antibodies The following antibodies were used for Western Blot and co-immunoprecipitation: p53 (DO-1 and CM-1) and MDM2 (4B2) were a kind gift from B.Vojtesek, p63 (4A4, Santa Cruz Biotechnology), p73 (5B429 mouse monoclonal, Abcam; rabbit polyclonal – gift from B.Vojtesek), HSP70 (SPA-812, Stressgen; 6B3, Cell Signaling), HSP40 (SPA-400, Stressgen), HSP90 (SPA-835, Stressgen), HSC70 (SPA-815, Stressgen), HA (3F10, Roche Molecular Biochemicals; 12CA5, Abcam), PARP (9542S, Cell Signaling), β-actin-HRP (AC-15, Sigma-Aldrich).

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Western Blot, Plasmid Preparation, Staining, FACS

    MDM2 dissociates HSP70 and HSP40 from the p53 R175H-TAp73α subcomplex and forms a three-body complex with p53 R175H and TAp73α ( A ) H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and MDM2, as indicated. 24 h post-transfection cells were lysed, protein complexes were immunoprecipitated with anti-p53 antibody and subjected to Western blot analysis. Lysates from cells lacking p53 R175H were used as a control of antibody specificity used for immunoprecipitation (Ctrl). ( B ) Double co-immunoprecipitation experiments (Two step Co-IP). H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and MDM2. After 24 h cellular proteins were cross-linked and first co-immunoprecipitation (1) with anti-p53 antibody was carried out. Protein complexes were eluted from beads (Cell lysates 2) and second co-immunoprecipitation (2) with anti-MDM2 antibody was performed. Laemmli buffer was supplemented with DTT to reverse cross-linking reaction. Lysates from cells transfected with plasmids encoding TAp73α and MDM2 but not p53 R175H were used as a control of the specificity of anti-p53 antibody. ( C ) Two step Co-IP with endogenous p53 R175H and MDM2 in SKBR3 cell line. Cells were transfected with a plasmid encoding TAp73α and the procedure was carried out as in (B) (left panel). Endogenous three-body complexes formed by p53 R175H, TAp73α and MDM2 were immunoprecipitated from SKBR3 cell lysates with anti-p73 antibody (right panel). ( D ) SKBR3 cell were treated with increasing concentrations of Nutlin-3 (0, 5, 10, 20 μM). After 24 h cellular proteins were cross-linked using 0.25 mM DSP and co-immunoprecipitation with anti-p53 antibody was carried out.

    Journal: Oncotarget

    Article Title: Molecular chaperones in the acquisition of cancer cell chemoresistance with mutated TP53 and MDM2 up-regulation

    doi: 10.18632/oncotarget.18899

    Figure Lengend Snippet: MDM2 dissociates HSP70 and HSP40 from the p53 R175H-TAp73α subcomplex and forms a three-body complex with p53 R175H and TAp73α ( A ) H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and MDM2, as indicated. 24 h post-transfection cells were lysed, protein complexes were immunoprecipitated with anti-p53 antibody and subjected to Western blot analysis. Lysates from cells lacking p53 R175H were used as a control of antibody specificity used for immunoprecipitation (Ctrl). ( B ) Double co-immunoprecipitation experiments (Two step Co-IP). H1299 cells were transfected with plasmids encoding p53 R175H, TAp73α and MDM2. After 24 h cellular proteins were cross-linked and first co-immunoprecipitation (1) with anti-p53 antibody was carried out. Protein complexes were eluted from beads (Cell lysates 2) and second co-immunoprecipitation (2) with anti-MDM2 antibody was performed. Laemmli buffer was supplemented with DTT to reverse cross-linking reaction. Lysates from cells transfected with plasmids encoding TAp73α and MDM2 but not p53 R175H were used as a control of the specificity of anti-p53 antibody. ( C ) Two step Co-IP with endogenous p53 R175H and MDM2 in SKBR3 cell line. Cells were transfected with a plasmid encoding TAp73α and the procedure was carried out as in (B) (left panel). Endogenous three-body complexes formed by p53 R175H, TAp73α and MDM2 were immunoprecipitated from SKBR3 cell lysates with anti-p73 antibody (right panel). ( D ) SKBR3 cell were treated with increasing concentrations of Nutlin-3 (0, 5, 10, 20 μM). After 24 h cellular proteins were cross-linked using 0.25 mM DSP and co-immunoprecipitation with anti-p53 antibody was carried out.

    Article Snippet: Antibodies The following antibodies were used for Western Blot and co-immunoprecipitation: p53 (DO-1 and CM-1) and MDM2 (4B2) were a kind gift from B.Vojtesek, p63 (4A4, Santa Cruz Biotechnology), p73 (5B429 mouse monoclonal, Abcam; rabbit polyclonal – gift from B.Vojtesek), HSP70 (SPA-812, Stressgen; 6B3, Cell Signaling), HSP40 (SPA-400, Stressgen), HSP90 (SPA-835, Stressgen), HSC70 (SPA-815, Stressgen), HA (3F10, Roche Molecular Biochemicals; 12CA5, Abcam), PARP (9542S, Cell Signaling), β-actin-HRP (AC-15, Sigma-Aldrich).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Plasmid Preparation

    HSP27 and HSP70 expression (WB) at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10) (3 wk study) Values are means ± SD; BFRE: n = 10 at Mid8 n = 9; LLE: n = 7.

    Journal: The Journal of Physiology

    Article Title: Blood flow restricted training leads to myocellular macrophage infiltration and upregulation of heat shock proteins, but no apparent muscle damage

    doi: 10.1113/JP273907

    Figure Lengend Snippet: HSP27 and HSP70 expression (WB) at baseline (Pre), 8 days into the training intervention (Mid8), and 3 and 10 days after cessation of training (Post3 and Post10) (3 wk study) Values are means ± SD; BFRE: n = 10 at Mid8 n = 9; LLE: n = 7.

    Article Snippet: Then, membranes were blocked at room temp. for 30 min in 5% non‐fat milk (170–6404, BR), after which they were incubated with primary antibodies: anti‐HSP27 (SPA‐803, StressGen (SG), San Diego, CA, USA; 1:1000), anti‐HSP70 (SPA‐810, SG; 1:1000) and GAPDH (D16H11, Cell Signalling, Danvers, MA, USA; 1:1000) at 4°C overnight.

    Techniques: Expressing, Western Blot

    Activation state of caspase-3 in response to nitric oxide. INS832/13 cells were treated for 1 h with 1 m m DEANO or with DEANO followed by washing to remove the donor and an additional 5 h or for 5 h with camptothecin as a positive control for apoptosis.

    Journal: The Journal of Biological Chemistry

    Article Title: AMP-activated Protein Kinase Attenuates Nitric Oxide-induced ?-Cell Death *

    doi: 10.1074/jbc.M109.047365

    Figure Lengend Snippet: Activation state of caspase-3 in response to nitric oxide. INS832/13 cells were treated for 1 h with 1 m m DEANO or with DEANO followed by washing to remove the donor and an additional 5 h or for 5 h with camptothecin as a positive control for apoptosis.

    Article Snippet: Phospho-Thr172-AMPK, phospho-Ser79-ACC, phospho-Ser473-Akt, phospho-Ser51-eIF2α, total AMPK, cleaved caspase-3, cleaved poly-(ADP-ribose) polymerase (PARP), Bcl2, BclXL (Cell Signaling, Danvers, MA), HSP90, HSP70, (Stressgen, Victoria, BC, Canada), CHOP/GADD153 (Santa Cruz Biotechnology, Santa Cruz, CA), GAPDH (Ambion,) horseradish peroxidase-conjugated donkey anti-rabbit and donkey anti-mouse were from Jackson Immunoresearch Laboratories, Inc (West Grove, PA).

    Techniques: Activation Assay, Positive Control

    AICAR promotes recovery of aconitase activity and attenuates caspase-3 activation. INS832/13 cells were treated the indicated concentration of AICAR for 1 h. The cells were isolated and phosphorylated ACC, AMPK, and total AMPKα were determined

    Journal: The Journal of Biological Chemistry

    Article Title: AMP-activated Protein Kinase Attenuates Nitric Oxide-induced ?-Cell Death *

    doi: 10.1074/jbc.M109.047365

    Figure Lengend Snippet: AICAR promotes recovery of aconitase activity and attenuates caspase-3 activation. INS832/13 cells were treated the indicated concentration of AICAR for 1 h. The cells were isolated and phosphorylated ACC, AMPK, and total AMPKα were determined

    Article Snippet: Phospho-Thr172-AMPK, phospho-Ser79-ACC, phospho-Ser473-Akt, phospho-Ser51-eIF2α, total AMPK, cleaved caspase-3, cleaved poly-(ADP-ribose) polymerase (PARP), Bcl2, BclXL (Cell Signaling, Danvers, MA), HSP90, HSP70, (Stressgen, Victoria, BC, Canada), CHOP/GADD153 (Santa Cruz Biotechnology, Santa Cruz, CA), GAPDH (Ambion,) horseradish peroxidase-conjugated donkey anti-rabbit and donkey anti-mouse were from Jackson Immunoresearch Laboratories, Inc (West Grove, PA).

    Techniques: Activity Assay, Activation Assay, Concentration Assay, Isolation

    Inhibition of AMPK attenuates recovery of aconitase activity and promotes caspase-3 activation during recovery. INS832/13 cells were transduced with increasing amounts of adenovirus expressing dominant negative or constitutively active AMPK mutants. The

    Journal: The Journal of Biological Chemistry

    Article Title: AMP-activated Protein Kinase Attenuates Nitric Oxide-induced ?-Cell Death *

    doi: 10.1074/jbc.M109.047365

    Figure Lengend Snippet: Inhibition of AMPK attenuates recovery of aconitase activity and promotes caspase-3 activation during recovery. INS832/13 cells were transduced with increasing amounts of adenovirus expressing dominant negative or constitutively active AMPK mutants. The

    Article Snippet: Phospho-Thr172-AMPK, phospho-Ser79-ACC, phospho-Ser473-Akt, phospho-Ser51-eIF2α, total AMPK, cleaved caspase-3, cleaved poly-(ADP-ribose) polymerase (PARP), Bcl2, BclXL (Cell Signaling, Danvers, MA), HSP90, HSP70, (Stressgen, Victoria, BC, Canada), CHOP/GADD153 (Santa Cruz Biotechnology, Santa Cruz, CA), GAPDH (Ambion,) horseradish peroxidase-conjugated donkey anti-rabbit and donkey anti-mouse were from Jackson Immunoresearch Laboratories, Inc (West Grove, PA).

    Techniques: Inhibition, Activity Assay, Activation Assay, Transduction, Expressing, Dominant Negative Mutation

    Double immunostaining confocal microscopy of HSP70 and Iba-1 or GFAP in P7 brain sections after HI. Brain sections were from P7 rats subjected to HI followed by 4 h of recovery (P7 4hR). Upper: HSP70 immunostaining (left, green) and microglia marker Iba-1 immunostaining (middle, red) are overlapped each other (right). Arrows point to immunopositive microglia. Lower: HSP70 immunostaining (left, green) and astrocyte marker GFAP immunostaining (middle, red) are not overlapped each other (right). Arrows point to immunopositive microglia. Arrowheads indicate astrocytes. Scale bar = 20 μm.

    Journal: Neurobiology of disease

    Article Title: Development-Dependent Regulation of Molecular Chaperones after Hypoxia-Ischemia

    doi: 10.1016/j.nbd.2015.06.001

    Figure Lengend Snippet: Double immunostaining confocal microscopy of HSP70 and Iba-1 or GFAP in P7 brain sections after HI. Brain sections were from P7 rats subjected to HI followed by 4 h of recovery (P7 4hR). Upper: HSP70 immunostaining (left, green) and microglia marker Iba-1 immunostaining (middle, red) are overlapped each other (right). Arrows point to immunopositive microglia. Lower: HSP70 immunostaining (left, green) and astrocyte marker GFAP immunostaining (middle, red) are not overlapped each other (right). Arrows point to immunopositive microglia. Arrowheads indicate astrocytes. Scale bar = 20 μm.

    Article Snippet: Antibodies to Hsp40, Hsp70, Hsp90, Grp78, Grp94, calnexin, and PDI were purchased from Stressgen Biotechnologies, Inc. (San Diego, CA).

    Techniques: Double Immunostaining, Confocal Microscopy, Immunostaining, Marker

    Expression of HSP70 after HI. Brain sections or tissue samples were from P7 or P26 sham-operated control rats and rats subjected to HI followed by 0.5, 4 and 24 h of recovery. A-left panels: Autoradiographs of in situ hybridization of hsp70 mRNA. Arrowheads point to the HI ipsilateral hemispheres. Arrows indicate the P26 contralateral CA1 area. The drawing areas indicate the ipsilateral hemisphere; A-right panels: Quantitative analysis of the hsp70 mRNA levels in the entire striatal level HI hemisphere (upper, Str-level Hemi) and the entire hippocampal level HI hemisphere (lower, Hip-level Hemi) of P7 (open bar) and P26 (black bar) brain sections, as delineated in the 30 min brain sections of Fig. 1A-left panels. B-left panels: Western blotting of HSP70 protein and beta-actin in tissue homogenates. Molecular size in kDa is indicated on the right; B-right panels: Quantitative analysis of the HSP70 protein level in P7 (open bar) and P26 (black bar) brain tissue samples. The HSP70 data were normalized with beta-actin loading control. The grey density value data are mean ± standard error (SEM) (n = 4). One-way ANOVA followed by Tukey’s post-hoc test. *denotes p

    Journal: Neurobiology of disease

    Article Title: Development-Dependent Regulation of Molecular Chaperones after Hypoxia-Ischemia

    doi: 10.1016/j.nbd.2015.06.001

    Figure Lengend Snippet: Expression of HSP70 after HI. Brain sections or tissue samples were from P7 or P26 sham-operated control rats and rats subjected to HI followed by 0.5, 4 and 24 h of recovery. A-left panels: Autoradiographs of in situ hybridization of hsp70 mRNA. Arrowheads point to the HI ipsilateral hemispheres. Arrows indicate the P26 contralateral CA1 area. The drawing areas indicate the ipsilateral hemisphere; A-right panels: Quantitative analysis of the hsp70 mRNA levels in the entire striatal level HI hemisphere (upper, Str-level Hemi) and the entire hippocampal level HI hemisphere (lower, Hip-level Hemi) of P7 (open bar) and P26 (black bar) brain sections, as delineated in the 30 min brain sections of Fig. 1A-left panels. B-left panels: Western blotting of HSP70 protein and beta-actin in tissue homogenates. Molecular size in kDa is indicated on the right; B-right panels: Quantitative analysis of the HSP70 protein level in P7 (open bar) and P26 (black bar) brain tissue samples. The HSP70 data were normalized with beta-actin loading control. The grey density value data are mean ± standard error (SEM) (n = 4). One-way ANOVA followed by Tukey’s post-hoc test. *denotes p

    Article Snippet: Antibodies to Hsp40, Hsp70, Hsp90, Grp78, Grp94, calnexin, and PDI were purchased from Stressgen Biotechnologies, Inc. (San Diego, CA).

    Techniques: Expressing, In Situ Hybridization, Western Blot

    Double immunostaining confocal microscopy of HSF1 (left panels, red) and HSP70 (right panels, green) of P26 brain sections (A and B) and of P7 brain sections (C and D) . Brain sections were from sham-operated control rats and rats subjected to HI followed by 0.5, 4 and 24 h of recovery. Three brain sections from three different rats in each experimental group were used in these confocal microscopic analyses. The results were reproducible. Arrows point to neuronal nuclei ( A and C ); Arrowheads indicate neuronal HSP70 immunostaining (B) ; Double-arrows point to non-neuronal HSP70 immunostaining (D) . Scale bar = 20 μm.

    Journal: Neurobiology of disease

    Article Title: Development-Dependent Regulation of Molecular Chaperones after Hypoxia-Ischemia

    doi: 10.1016/j.nbd.2015.06.001

    Figure Lengend Snippet: Double immunostaining confocal microscopy of HSF1 (left panels, red) and HSP70 (right panels, green) of P26 brain sections (A and B) and of P7 brain sections (C and D) . Brain sections were from sham-operated control rats and rats subjected to HI followed by 0.5, 4 and 24 h of recovery. Three brain sections from three different rats in each experimental group were used in these confocal microscopic analyses. The results were reproducible. Arrows point to neuronal nuclei ( A and C ); Arrowheads indicate neuronal HSP70 immunostaining (B) ; Double-arrows point to non-neuronal HSP70 immunostaining (D) . Scale bar = 20 μm.

    Article Snippet: Antibodies to Hsp40, Hsp70, Hsp90, Grp78, Grp94, calnexin, and PDI were purchased from Stressgen Biotechnologies, Inc. (San Diego, CA).

    Techniques: Double Immunostaining, Confocal Microscopy, Immunostaining

    Protein levels of Hsp90, Hsp70, and Hsp27. Control and/or heat shocked utricles from CBA mice, Hsp70 -overexpressing mice, and wild-type littermates were collected 6 h after heat shock. Protein lysates were subjected to SDS-PAGE and then transferred

    Journal:

    Article Title: Hsp70 Inhibits Aminoglycoside-Induced Hair Cell Death and is Necessary for the Protective Effect of Heat Shock

    doi: 10.1007/s10162-008-0122-2

    Figure Lengend Snippet: Protein levels of Hsp90, Hsp70, and Hsp27. Control and/or heat shocked utricles from CBA mice, Hsp70 -overexpressing mice, and wild-type littermates were collected 6 h after heat shock. Protein lysates were subjected to SDS-PAGE and then transferred

    Article Snippet: Primary antibodies included anti-Hsp90 (Cell Signaling Technology no. 4874, Danvers, MA, USA; 1:1,000), anti-Hsp70 (Stressgen no. SPA-810; 1:1,000), anti-Hsp27 (Upstate no. 06-517, Lake Placid, NY, USA; 1:1,000), and anti-actin (Sigma no. A 2066; 1:1,000).

    Techniques: Crocin Bleaching Assay, Mouse Assay, SDS Page

    EGCG modifies the interaction of hsp90 with cochaperones, and leads to the degradation of the hsp90 client proteins ErbB2, Raf-1 and phospho-AKT. A. Hepa cytosol was incubated with DMSO or 200μM EGCG (E) for 1h at RT and immunoprecipitated with

    Journal: Biochemistry

    Article Title: (-)-EPIGALLOCATECHIN-3-GALLATE IS A NOVEL HSP90 INHIBITOR †

    doi: 10.1021/bi801637q

    Figure Lengend Snippet: EGCG modifies the interaction of hsp90 with cochaperones, and leads to the degradation of the hsp90 client proteins ErbB2, Raf-1 and phospho-AKT. A. Hepa cytosol was incubated with DMSO or 200μM EGCG (E) for 1h at RT and immunoprecipitated with

    Article Snippet: Following SDS-PAGE, the resolved bands were transferred to PVDF membrane (Millipore, Billerica, MA) and blotted with antibodies against AhR (RPT-1), XAP2, p23, Cyp40, N-terminal hsp90 (PA3-013) (Affinity BioReagents, Golden, CO), C-terminal hsp90 (AC88), hsp70 (Stressgen), AKT, phospho-AKT (Cell Signaling Technology, Danvers, MA), ErbB2 or Raf-1 (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Incubation, Immunoprecipitation

    Effects of S100 proteins on the CHIP-client protein interactions in vitro . A–E , upper panel , GST-CHIP (20 μg), S100 proteins (20 μg each) and appropriate client proteins were mixed with glutathione-Sepharose 4B in the presence of CaCl 2 or EGTA. Control assay ( CHIP− ) was conducted using GST (20 μg). S100 proteins used were as follows: S100A2, S100A6, S100P, S100A12, and control (−). The client proteins were as follows: purified 20 μg of Hsp70 ( A ), 40 μg of Hsp90 ( B ), 20 μg of Smad1 ( C ), 20 μg of HSF1 ( D ), and 20 μg of UbcH5a ( E ). Details of the GST pulldown assay are described under “Experimental Procedures.” The resulting samples were subjected to SDS-PAGE and visualized by Western blotting ( WB) using the indicated antibodies. Lower panel , the binding levels of these client proteins to CHIP without S100 proteins ( lane 1 ) were designated as 100% ( Control ), and the relative binding levels ( % of Control ) were plotted. The error bars represent the S.E. with n = 3.

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein) *

    doi: 10.1074/jbc.M112.436758

    Figure Lengend Snippet: Effects of S100 proteins on the CHIP-client protein interactions in vitro . A–E , upper panel , GST-CHIP (20 μg), S100 proteins (20 μg each) and appropriate client proteins were mixed with glutathione-Sepharose 4B in the presence of CaCl 2 or EGTA. Control assay ( CHIP− ) was conducted using GST (20 μg). S100 proteins used were as follows: S100A2, S100A6, S100P, S100A12, and control (−). The client proteins were as follows: purified 20 μg of Hsp70 ( A ), 40 μg of Hsp90 ( B ), 20 μg of Smad1 ( C ), 20 μg of HSF1 ( D ), and 20 μg of UbcH5a ( E ). Details of the GST pulldown assay are described under “Experimental Procedures.” The resulting samples were subjected to SDS-PAGE and visualized by Western blotting ( WB) using the indicated antibodies. Lower panel , the binding levels of these client proteins to CHIP without S100 proteins ( lane 1 ) were designated as 100% ( Control ), and the relative binding levels ( % of Control ) were plotted. The error bars represent the S.E. with n = 3.

    Article Snippet: The following antibodies were used in this study: anti-Smad1 (R & D Systems), anti-S100A2 (R & D Systems), anti-S100P (R & D Systems), anti-S100A12 (R & D Systems), anti-HSF1 (StressGen Biotechnologies), anti-Hsp70 (StressGen Biotechnologies), anti-Hsp90 (StressGen Biotechnologies), anti-β-actin (Santa Cruz Biotechnology), anti-CHIP (Santa Cruz Biotechnology), anti-CHIP (clone EPR4448, Epitomics Inc.), anti-S100A6 (Epitomics Inc.), anti-UbcH5 (Boston Biochem), anti-FLAG (Sigma Aldrich), anti-p53 (clone DO-1, Santa Cruz Biotechnology), and anti-ubiquitin (clone FK2, BIOMOL International).

    Techniques: Chromatin Immunoprecipitation, In Vitro, Control Assay, Purification, GST Pulldown Assay, SDS Page, Western Blot, Binding Assay

    FN-Integrin signaling inhibitor G RGD SP and ERK1/2 kinase inhibitor PD98059 attenuate GLN-mediated activation of HSF-1. A) IEC-6 cells were treated with 0 mM or 10 mM GLN with or without 1 h prior PD98059 or G RGD SP treatment. Representative Western blot from 3 independent experiments shows [S(P) 303 ]HSF-1 and total HSF-1 levels. B) Densitometric analysis of HSF-1 phosphorylation at serine 303 was measured as mean fold changes relative to total HSF-1±SEM (n = 3). C) Total HSF-1 levels were measured after 3 h recovery after non-lethal HS. Results are displayed as fold change±SEM ratioed to HS 0 mM GLN groups (n = 3).

    Journal: PLoS ONE

    Article Title: Fibronectin-Integrin Signaling Is Required for L-Glutamine's Protection against Gut Injury

    doi: 10.1371/journal.pone.0050185

    Figure Lengend Snippet: FN-Integrin signaling inhibitor G RGD SP and ERK1/2 kinase inhibitor PD98059 attenuate GLN-mediated activation of HSF-1. A) IEC-6 cells were treated with 0 mM or 10 mM GLN with or without 1 h prior PD98059 or G RGD SP treatment. Representative Western blot from 3 independent experiments shows [S(P) 303 ]HSF-1 and total HSF-1 levels. B) Densitometric analysis of HSF-1 phosphorylation at serine 303 was measured as mean fold changes relative to total HSF-1±SEM (n = 3). C) Total HSF-1 levels were measured after 3 h recovery after non-lethal HS. Results are displayed as fold change±SEM ratioed to HS 0 mM GLN groups (n = 3).

    Article Snippet: Primary antibodies against HSP32 and HSP70 (1∶10,000 dilution) (StressGen, Victoria, BC, Canada) and FN (1∶10,000), or against total ERK1/2, [T(P)202 /Y(P)204 ]ERK1/2, total HSF-1, [S(P)303 ]HSF-1, cleaved caspase-3, cleaved PARP, bax, and bcl-2 (1∶1,000) (Cell signaling, Danvers, MA) were used.

    Techniques: Activation Assay, Western Blot

    ERK1/2 activation is involved in GLN’s protective mechanism and attenuates after FN-Integrin pathway inhibition. A) IEC-6 cells were treated with different concentrations of GLN (0, 2, 10, and 20 mM) with or without 1 h prior PD98059 treatment. Cell survival was measured via MTS assay. Results are shown as mean±SEM (n = 3). B) [T(P) 202 /Y(P) 204 ]ERK1/2 and total ERK1/2 levels were determined by Western blot analysis after basal and stressed 43°C conditions without recovery. ERK1/2 activation is shown as mean fold change relative to total ERK1/2±SEM and ratioed to 0 mM GLN (n = 3).

    Journal: PLoS ONE

    Article Title: Fibronectin-Integrin Signaling Is Required for L-Glutamine's Protection against Gut Injury

    doi: 10.1371/journal.pone.0050185

    Figure Lengend Snippet: ERK1/2 activation is involved in GLN’s protective mechanism and attenuates after FN-Integrin pathway inhibition. A) IEC-6 cells were treated with different concentrations of GLN (0, 2, 10, and 20 mM) with or without 1 h prior PD98059 treatment. Cell survival was measured via MTS assay. Results are shown as mean±SEM (n = 3). B) [T(P) 202 /Y(P) 204 ]ERK1/2 and total ERK1/2 levels were determined by Western blot analysis after basal and stressed 43°C conditions without recovery. ERK1/2 activation is shown as mean fold change relative to total ERK1/2±SEM and ratioed to 0 mM GLN (n = 3).

    Article Snippet: Primary antibodies against HSP32 and HSP70 (1∶10,000 dilution) (StressGen, Victoria, BC, Canada) and FN (1∶10,000), or against total ERK1/2, [T(P)202 /Y(P)204 ]ERK1/2, total HSF-1, [S(P)303 ]HSF-1, cleaved caspase-3, cleaved PARP, bax, and bcl-2 (1∶1,000) (Cell signaling, Danvers, MA) were used.

    Techniques: Activation Assay, Inhibition, MTS Assay, Western Blot

    Proposed working model. GLN is protective in the intestine by preventing FN degradation after thermal injury, as well as by activating the protective FN-Integrin signaling pathway. GLN phosphorylates ERK1/2 via the FN-Integrin pathway leading to HSF-1 activation, which enhances HSP expression to prevent apoptosis.

    Journal: PLoS ONE

    Article Title: Fibronectin-Integrin Signaling Is Required for L-Glutamine's Protection against Gut Injury

    doi: 10.1371/journal.pone.0050185

    Figure Lengend Snippet: Proposed working model. GLN is protective in the intestine by preventing FN degradation after thermal injury, as well as by activating the protective FN-Integrin signaling pathway. GLN phosphorylates ERK1/2 via the FN-Integrin pathway leading to HSF-1 activation, which enhances HSP expression to prevent apoptosis.

    Article Snippet: Primary antibodies against HSP32 and HSP70 (1∶10,000 dilution) (StressGen, Victoria, BC, Canada) and FN (1∶10,000), or against total ERK1/2, [T(P)202 /Y(P)204 ]ERK1/2, total HSF-1, [S(P)303 ]HSF-1, cleaved caspase-3, cleaved PARP, bax, and bcl-2 (1∶1,000) (Cell signaling, Danvers, MA) were used.

    Techniques: Activation Assay, Expressing

    Endoscopic and histological grading of DSS colitis in WT and Hsp70 TG mice. WT and Hsp70 UTR-less TG mice were given 3% DSS for 7 days and then given tap water for another 3 days before analysis. A : colonoscopy was performed before and after DSS

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Inflammation-induced, 3?UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis

    doi: 10.1152/ajpgi.00027.2009

    Figure Lengend Snippet: Endoscopic and histological grading of DSS colitis in WT and Hsp70 TG mice. WT and Hsp70 UTR-less TG mice were given 3% DSS for 7 days and then given tap water for another 3 days before analysis. A : colonoscopy was performed before and after DSS

    Article Snippet: Blots were incubated overnight at 4°C in primary antibodies for Hsp70 (SPA810; StressGen/Assay Designs, Ann Arbor, MI), Hsc70 (SPA815, StressGen), and cytokeratin 18 (18–0158; Zymed Laboratory, South San Francisco, CA).

    Techniques: Mouse Assay

    Protein expression of UTR-less Hsp70 mRNA is resistant to DSS-induced inflammation. WT and Hsp70 UTR-less TG mice were given 3% DSS for 7 days and then euthanized. Mice given normal tap water were euthanized as controls. Colonic mucosa of transverse

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Inflammation-induced, 3?UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis

    doi: 10.1152/ajpgi.00027.2009

    Figure Lengend Snippet: Protein expression of UTR-less Hsp70 mRNA is resistant to DSS-induced inflammation. WT and Hsp70 UTR-less TG mice were given 3% DSS for 7 days and then euthanized. Mice given normal tap water were euthanized as controls. Colonic mucosa of transverse

    Article Snippet: Blots were incubated overnight at 4°C in primary antibodies for Hsp70 (SPA810; StressGen/Assay Designs, Ann Arbor, MI), Hsc70 (SPA815, StressGen), and cytokeratin 18 (18–0158; Zymed Laboratory, South San Francisco, CA).

    Techniques: Expressing, Mouse Assay

    Hsp70 knockout (KO) mice are more susceptible to DSS treatment than wild-type (WT) mice. Hsp70 KO mice (Hsp 70.1 −/− and 70.3 −/− ) and WT littermates were given DSS in drinking water. A : survival rate of WT and Hsp70 KO mice

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Inflammation-induced, 3?UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis

    doi: 10.1152/ajpgi.00027.2009

    Figure Lengend Snippet: Hsp70 knockout (KO) mice are more susceptible to DSS treatment than wild-type (WT) mice. Hsp70 KO mice (Hsp 70.1 −/− and 70.3 −/− ) and WT littermates were given DSS in drinking water. A : survival rate of WT and Hsp70 KO mice

    Article Snippet: Blots were incubated overnight at 4°C in primary antibodies for Hsp70 (SPA810; StressGen/Assay Designs, Ann Arbor, MI), Hsc70 (SPA815, StressGen), and cytokeratin 18 (18–0158; Zymed Laboratory, South San Francisco, CA).

    Techniques: Knock-Out, Mouse Assay

    Colonic heat shock protein 70 (Hsp70) decreases in dextran sodium sulfate (DSS)-induced inflammation. C57BL/6 mice were given 3% wt/vol DSS for up to 7 days in their drinking water. On days 0 , 2 , 3 , 4 , 5 , 6 , and 7 , DSS-treated and control mice

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Inflammation-induced, 3?UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis

    doi: 10.1152/ajpgi.00027.2009

    Figure Lengend Snippet: Colonic heat shock protein 70 (Hsp70) decreases in dextran sodium sulfate (DSS)-induced inflammation. C57BL/6 mice were given 3% wt/vol DSS for up to 7 days in their drinking water. On days 0 , 2 , 3 , 4 , 5 , 6 , and 7 , DSS-treated and control mice

    Article Snippet: Blots were incubated overnight at 4°C in primary antibodies for Hsp70 (SPA810; StressGen/Assay Designs, Ann Arbor, MI), Hsc70 (SPA815, StressGen), and cytokeratin 18 (18–0158; Zymed Laboratory, South San Francisco, CA).

    Techniques: Mouse Assay

    Hsp70 3′ untranslated regions (UTRs) participate in translational inhibition induced by IFN-γ and TNF-α in young adult mouse colonic epithelial cells (YAMC) cells. A : schematic of constructs expressing chimeric luciferase transcripts

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Inflammation-induced, 3?UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis

    doi: 10.1152/ajpgi.00027.2009

    Figure Lengend Snippet: Hsp70 3′ untranslated regions (UTRs) participate in translational inhibition induced by IFN-γ and TNF-α in young adult mouse colonic epithelial cells (YAMC) cells. A : schematic of constructs expressing chimeric luciferase transcripts

    Article Snippet: Blots were incubated overnight at 4°C in primary antibodies for Hsp70 (SPA810; StressGen/Assay Designs, Ann Arbor, MI), Hsc70 (SPA815, StressGen), and cytokeratin 18 (18–0158; Zymed Laboratory, South San Francisco, CA).

    Techniques: Inhibition, Construct, Expressing, Luciferase

    Intestine-specific Hsp70 expression from “UTR-less” Hsp70 mRNA in villin-Hsp70 transgenic (TG) mice. A : schematic of targeting transgene construct. The 11.1-kbp fragment, comprising the villin promoter followed by the human Hsp70 protein

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Inflammation-induced, 3?UTR-dependent translational inhibition of Hsp70 mRNA impairs intestinal homeostasis

    doi: 10.1152/ajpgi.00027.2009

    Figure Lengend Snippet: Intestine-specific Hsp70 expression from “UTR-less” Hsp70 mRNA in villin-Hsp70 transgenic (TG) mice. A : schematic of targeting transgene construct. The 11.1-kbp fragment, comprising the villin promoter followed by the human Hsp70 protein

    Article Snippet: Blots were incubated overnight at 4°C in primary antibodies for Hsp70 (SPA810; StressGen/Assay Designs, Ann Arbor, MI), Hsc70 (SPA815, StressGen), and cytokeratin 18 (18–0158; Zymed Laboratory, South San Francisco, CA).

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Construct

    Identification of miRNAs involved in gefitinib-dependent suppression of expression of HSP70. A549 cells were incubated with the indicated concentration of gefitinib for 3 h (A). A549 cells were transfected with siRNA for Dicer1 (siDicer1) or non-specific siRNA (ns) and after 24 h were incubated with the indicated concentration of gefitinib for 3 h (B). A549 cells were transfected with the indicated amount ( µg/well) of miRNA mimic RNA fragments for 24 h (C, E). The RNA (A, B, E) and protein (C) expression was monitored and expressed as described in the legend of Fig. 1 . The RUN44 non-coding RNA was used for normalization (A, B, E). The intensities of the HSP70 bands were determined and are expressed relative to the control (one of the gels is shown in C) (D). Values shown are mean ± S.D. ( n = 3). ** P

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Identification of miRNAs involved in gefitinib-dependent suppression of expression of HSP70. A549 cells were incubated with the indicated concentration of gefitinib for 3 h (A). A549 cells were transfected with siRNA for Dicer1 (siDicer1) or non-specific siRNA (ns) and after 24 h were incubated with the indicated concentration of gefitinib for 3 h (B). A549 cells were transfected with the indicated amount ( µg/well) of miRNA mimic RNA fragments for 24 h (C, E). The RNA (A, B, E) and protein (C) expression was monitored and expressed as described in the legend of Fig. 1 . The RUN44 non-coding RNA was used for normalization (A, B, E). The intensities of the HSP70 bands were determined and are expressed relative to the control (one of the gels is shown in C) (D). Values shown are mean ± S.D. ( n = 3). ** P

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Expressing, Incubation, Concentration Assay, Transfection

    Effect of GGA on gefitinib-dependent exacerbation of pulmonary fibrosis and pulmonary expression of HSP70. Wild-type mice were orally administered gefitinib (200 mg/kg) and/or GGA (200 mg/kg) once per day for 3 days (from day 0 to day 2), then treated once only with 1 mg/kg bleomycin (BLM) (day 3). Mice were then orally administered gefitinib (200 mg/kg) and/or GGA (200 mg/kg) once per 2 days for 14 days (from day 3 to day 17). Sections of pulmonary tissue were prepared at day 17 and subjected to histological examination (H E staining (A) or Masson's trichrome staining (B)) (scale bar, 50 µm). The pulmonary hydroxyproline levels were determined at day 17 (C). Total protein was extracted from pulmonary tissues at day 17 and analyzed and expressed as described in the legend of Fig. 6 (D, E). Values are mean ± S.E.M. ** P

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Effect of GGA on gefitinib-dependent exacerbation of pulmonary fibrosis and pulmonary expression of HSP70. Wild-type mice were orally administered gefitinib (200 mg/kg) and/or GGA (200 mg/kg) once per day for 3 days (from day 0 to day 2), then treated once only with 1 mg/kg bleomycin (BLM) (day 3). Mice were then orally administered gefitinib (200 mg/kg) and/or GGA (200 mg/kg) once per 2 days for 14 days (from day 3 to day 17). Sections of pulmonary tissue were prepared at day 17 and subjected to histological examination (H E staining (A) or Masson's trichrome staining (B)) (scale bar, 50 µm). The pulmonary hydroxyproline levels were determined at day 17 (C). Total protein was extracted from pulmonary tissues at day 17 and analyzed and expressed as described in the legend of Fig. 6 (D, E). Values are mean ± S.E.M. ** P

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Expressing, Mouse Assay, Staining

    Involvement of inhibition of EGFR and JNK in gefitinib-dependent suppression of expression of HSP70. A549 (A-C) and H1975 and PC9 (A) cells were incubated with the indicated concentration of gefitinib (A, B) or SP600125 (B) for 24 h. Protein expression was monitored and is expressed as described in the legend of Fig. 1 .

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Involvement of inhibition of EGFR and JNK in gefitinib-dependent suppression of expression of HSP70. A549 (A-C) and H1975 and PC9 (A) cells were incubated with the indicated concentration of gefitinib (A, B) or SP600125 (B) for 24 h. Protein expression was monitored and is expressed as described in the legend of Fig. 1 .

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Inhibition, Expressing, Incubation, Concentration Assay

    Suppression of expression of HSPs by gefitinib. A549 cells were incubated with the indicated concentration of gefitinib (A, C) or erlotinib (B) for 24 h (A, B) or 12 h (C). Whole-cell extracts were analyzed by immunoblotting with an antibody against HSP70, HSP90, HSP27, HSP47, HSP60 or actin (A, B). Total RNA was extracted and subjected to real-time RT-PCR using a specific primer set for each gene. Values were normalized to the actin gene, expressed relative to the control sample (C). Values shown are mean ± S.D. ( n = 3). ** P

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Suppression of expression of HSPs by gefitinib. A549 cells were incubated with the indicated concentration of gefitinib (A, C) or erlotinib (B) for 24 h (A, B) or 12 h (C). Whole-cell extracts were analyzed by immunoblotting with an antibody against HSP70, HSP90, HSP27, HSP47, HSP60 or actin (A, B). Total RNA was extracted and subjected to real-time RT-PCR using a specific primer set for each gene. Values were normalized to the actin gene, expressed relative to the control sample (C). Values shown are mean ± S.D. ( n = 3). ** P

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Expressing, Incubation, Concentration Assay, Quantitative RT-PCR

    Effect of gefitinib on bleomycin-induced pulmonary fibrosis and pulmonary expression of HSP70. Wild-type mice (A, B, E, G, H) and transgenic mice expressing HSP70 (C, D, F, G, J) were orally administered gefitinib (200 mg/kg) or vehicle once per day for 3 days (from day 0 to day 2), then treated once only with or without 1 mg/kg (for wild-type mice) or 2 mg/kg (for the transgenic mice) bleomycin (BLM) (day 3). Mice were then orally administered gefitinib (200 mg/kg) or vehicle once per 2 days for 14 days (from day 3 to day 17). Sections of pulmonary tissue were prepared at day 17 and subjected to histological examination (H E staining (A, C) or Masson's trichrome staining (B, D)) (scale bar, 50 µm) (A-D). Pulmonary hydroxyproline levels at day 17 were determined (E, F). Total protein was extracted from pulmonary tissues at day 17 and analyzed by immunoblotting with an antibody against HSP70 or actin (G, H, J). The intensities of the HSP70 bands were determined (one of the gels is shown in H) and are expressed relative to the control (I). Values are mean ± S.E.M. * P

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Effect of gefitinib on bleomycin-induced pulmonary fibrosis and pulmonary expression of HSP70. Wild-type mice (A, B, E, G, H) and transgenic mice expressing HSP70 (C, D, F, G, J) were orally administered gefitinib (200 mg/kg) or vehicle once per day for 3 days (from day 0 to day 2), then treated once only with or without 1 mg/kg (for wild-type mice) or 2 mg/kg (for the transgenic mice) bleomycin (BLM) (day 3). Mice were then orally administered gefitinib (200 mg/kg) or vehicle once per 2 days for 14 days (from day 3 to day 17). Sections of pulmonary tissue were prepared at day 17 and subjected to histological examination (H E staining (A, C) or Masson's trichrome staining (B, D)) (scale bar, 50 µm) (A-D). Pulmonary hydroxyproline levels at day 17 were determined (E, F). Total protein was extracted from pulmonary tissues at day 17 and analyzed by immunoblotting with an antibody against HSP70 or actin (G, H, J). The intensities of the HSP70 bands were determined (one of the gels is shown in H) and are expressed relative to the control (I). Values are mean ± S.E.M. * P

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Expressing, Mouse Assay, Transgenic Assay, Staining

    Contribution of miRNA to gefitinib-dependent suppression of expression of HSP70. A549 cells were co-transfected with pRL-SV40 and pMIR/luc/ hsp70 3' UTR and cultured for 24 h. Cells were incubated with the indicated concentration of gefitinib for 24 h and luciferase reporter assay was done as described in the legend of Fig. 2 . The 100% value of the P. pyralis luciferase activity is 1.2×10 5 units (A). A549 cells were transfected with siRNA for Dicer1 (siDicer1) or non-specific siRNA (ns) (B-D). After 24 h, cells were incubated with the indicated concentration of gefitinib for 12 h (B), 24 h (C) or 8 h (D). The mRNA (B) and protein (C) expression was monitored and is expressed as described in the legend of Fig. 1 . Pulse-labelling experiments were performed as described in the legend of Fig. 2 (D). Values shown are mean ± S.D. ( n = 3). ** P

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Contribution of miRNA to gefitinib-dependent suppression of expression of HSP70. A549 cells were co-transfected with pRL-SV40 and pMIR/luc/ hsp70 3' UTR and cultured for 24 h. Cells were incubated with the indicated concentration of gefitinib for 24 h and luciferase reporter assay was done as described in the legend of Fig. 2 . The 100% value of the P. pyralis luciferase activity is 1.2×10 5 units (A). A549 cells were transfected with siRNA for Dicer1 (siDicer1) or non-specific siRNA (ns) (B-D). After 24 h, cells were incubated with the indicated concentration of gefitinib for 12 h (B), 24 h (C) or 8 h (D). The mRNA (B) and protein (C) expression was monitored and is expressed as described in the legend of Fig. 1 . Pulse-labelling experiments were performed as described in the legend of Fig. 2 (D). Values shown are mean ± S.D. ( n = 3). ** P

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Expressing, Transfection, Cell Culture, Incubation, Concentration Assay, Luciferase, Reporter Assay, Activity Assay

    Translational regulation of expression of HSP70 by gefitinib. A549 cells were co-transfected with pRL-SV40 (internal control plasmid carrying the R. reniformis luciferase gene) and a pGL-3 derivative (pGL-3/ hsp70 pro or pGL-3/HSE) and cultured for 24 h. Cells were incubated with the indicated concentration of gefitinib for 24 h and P. pyralis luciferase activity was measured and normalized for R. reniformis luciferase activity. The 100% value of the P. pyralis luciferase activity is 6.9×10 4 or 5.8×10 4 units for pGL-3/ hsp70 pro or pGL-3/HSE, respectively (A). A549 cells were pre-incubated with 1 µg/ml actinomycin D (ActD) (B) or 20 µg/ml cycloheximide (C) for 1 h and further incubated for 8 h (B) or 24 h (C) with the indicated concentration of gefitinib (B, C). The mRNA (B) and protein (C) expression was monitored and is expressed as described in the legend of Fig. 1 A549 cells were pulse-labelled for 15 min with [ 35 S]methionine and [ 35 S]cysteine (D, E). Before the pulse-labelling, cells were incubated with the indicated concentration of gefitinib for 8 h (D). Pulse-labelled proteins were chased with excess amounts of Non-radioactively labeled methionine and cysteine for the indicated period in the presence of the indicated concentration of gefitinib (E). Labelled proteins were extracted, immunoprecipitated with antibody against HSP70, subjected to SDS-PAGE and autoradiographed (D, E). Values are mean ± S.D. ( n = 3). ** P

    Journal: PLoS ONE

    Article Title: Suppression of Expression of Heat Shock Protein 70 by Gefitinib and Its Contribution to Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0027296

    Figure Lengend Snippet: Translational regulation of expression of HSP70 by gefitinib. A549 cells were co-transfected with pRL-SV40 (internal control plasmid carrying the R. reniformis luciferase gene) and a pGL-3 derivative (pGL-3/ hsp70 pro or pGL-3/HSE) and cultured for 24 h. Cells were incubated with the indicated concentration of gefitinib for 24 h and P. pyralis luciferase activity was measured and normalized for R. reniformis luciferase activity. The 100% value of the P. pyralis luciferase activity is 6.9×10 4 or 5.8×10 4 units for pGL-3/ hsp70 pro or pGL-3/HSE, respectively (A). A549 cells were pre-incubated with 1 µg/ml actinomycin D (ActD) (B) or 20 µg/ml cycloheximide (C) for 1 h and further incubated for 8 h (B) or 24 h (C) with the indicated concentration of gefitinib (B, C). The mRNA (B) and protein (C) expression was monitored and is expressed as described in the legend of Fig. 1 A549 cells were pulse-labelled for 15 min with [ 35 S]methionine and [ 35 S]cysteine (D, E). Before the pulse-labelling, cells were incubated with the indicated concentration of gefitinib for 8 h (D). Pulse-labelled proteins were chased with excess amounts of Non-radioactively labeled methionine and cysteine for the indicated period in the presence of the indicated concentration of gefitinib (E). Labelled proteins were extracted, immunoprecipitated with antibody against HSP70, subjected to SDS-PAGE and autoradiographed (D, E). Values are mean ± S.D. ( n = 3). ** P

    Article Snippet: Antibodies against actin and HSP70 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and BD Bioscience (San Francisco, CA), respectively.

    Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Cell Culture, Incubation, Concentration Assay, Activity Assay, Labeling, Immunoprecipitation, SDS Page

    Hsp90β (S226A/S255A) inhibits Apaf-1 oligomerization and caspase-9 recruitment. Recombinant Apaf-1 (0.4 μM) was mixed with catalytically inactive caspase-9 [C9 (C287A)] (0.8 μM) and recombinant Hsp90β (1 μM; S226E/S255E

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of Apoptosome Formation by Suppression of Hsp90? Phosphorylation in Tyrosine Kinase-Induced Leukemias ▿

    doi: 10.1128/MCB.00265-08

    Figure Lengend Snippet: Hsp90β (S226A/S255A) inhibits Apaf-1 oligomerization and caspase-9 recruitment. Recombinant Apaf-1 (0.4 μM) was mixed with catalytically inactive caspase-9 [C9 (C287A)] (0.8 μM) and recombinant Hsp90β (1 μM; S226E/S255E

    Article Snippet: Anti-caspase-9 (Neomarkers, Fremont, CA), anti-caspase-9 (mouse specific; Cell Signaling, Danvers, MA), anti-cleaved caspase-3, anti-caspase-3 (mouse specific), anti-cytochrome c , anti-Abl (BD Biosciences, San Diego, CA), anti-Apaf-1 (Anaspec, San Jose, CA; Alexis Biochemicals, San Diego, CA), anti-Hsp70 (Affinity Bioreagents, Golden, CO), anti-Hsp90 (Stressgen Bioreagents, Ann Arbor, MI; Upstate Biotechnology, Lake Placid, NY; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PDGFRβ (Upstate Biotechnology), anti-FLT3, antiactin (Santa Cruz Biotechnology), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, Eugene, OR), and anti-FLAG (Sigma, St. Louis, MO) were used for immunoblotting.

    Techniques: Recombinant

    The phosphorylation of Hsp90β controls its inhibitory effect on Apaf-1 oligomerization. (A) Recombinant human caspase-9 (C287A) and human Apaf-1 proteins were produced and purified from BL21(DE3) and Sf9 cells, respectively, as described in Materials

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of Apoptosome Formation by Suppression of Hsp90? Phosphorylation in Tyrosine Kinase-Induced Leukemias ▿

    doi: 10.1128/MCB.00265-08

    Figure Lengend Snippet: The phosphorylation of Hsp90β controls its inhibitory effect on Apaf-1 oligomerization. (A) Recombinant human caspase-9 (C287A) and human Apaf-1 proteins were produced and purified from BL21(DE3) and Sf9 cells, respectively, as described in Materials

    Article Snippet: Anti-caspase-9 (Neomarkers, Fremont, CA), anti-caspase-9 (mouse specific; Cell Signaling, Danvers, MA), anti-cleaved caspase-3, anti-caspase-3 (mouse specific), anti-cytochrome c , anti-Abl (BD Biosciences, San Diego, CA), anti-Apaf-1 (Anaspec, San Jose, CA; Alexis Biochemicals, San Diego, CA), anti-Hsp70 (Affinity Bioreagents, Golden, CO), anti-Hsp90 (Stressgen Bioreagents, Ann Arbor, MI; Upstate Biotechnology, Lake Placid, NY; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PDGFRβ (Upstate Biotechnology), anti-FLT3, antiactin (Santa Cruz Biotechnology), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, Eugene, OR), and anti-FLAG (Sigma, St. Louis, MO) were used for immunoblotting.

    Techniques: Recombinant, Produced, Purification

    Leukemogenic tyrosine kinases inhibit recruitment of caspase-9 to Apaf-1. (A) Cell lysates were prepared from control Ba/F3 cells or Ba/F3 cells expressing Bcr-Abl, FLT3/D835Y, or Tel-PDGFRβ and incubated with 0 or 2.5 ng/μl cytochrome

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of Apoptosome Formation by Suppression of Hsp90? Phosphorylation in Tyrosine Kinase-Induced Leukemias ▿

    doi: 10.1128/MCB.00265-08

    Figure Lengend Snippet: Leukemogenic tyrosine kinases inhibit recruitment of caspase-9 to Apaf-1. (A) Cell lysates were prepared from control Ba/F3 cells or Ba/F3 cells expressing Bcr-Abl, FLT3/D835Y, or Tel-PDGFRβ and incubated with 0 or 2.5 ng/μl cytochrome

    Article Snippet: Anti-caspase-9 (Neomarkers, Fremont, CA), anti-caspase-9 (mouse specific; Cell Signaling, Danvers, MA), anti-cleaved caspase-3, anti-caspase-3 (mouse specific), anti-cytochrome c , anti-Abl (BD Biosciences, San Diego, CA), anti-Apaf-1 (Anaspec, San Jose, CA; Alexis Biochemicals, San Diego, CA), anti-Hsp70 (Affinity Bioreagents, Golden, CO), anti-Hsp90 (Stressgen Bioreagents, Ann Arbor, MI; Upstate Biotechnology, Lake Placid, NY; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PDGFRβ (Upstate Biotechnology), anti-FLT3, antiactin (Santa Cruz Biotechnology), goat anti-rabbit Alexa Fluor 647 (Molecular Probes, Eugene, OR), and anti-FLAG (Sigma, St. Louis, MO) were used for immunoblotting.

    Techniques: Expressing, Incubation

    Effect of HSP90 inhibition on downstream signaling. BON1, NCI-H727 and GOT1 cells were treated with increasing concentrations (1–100 nM) of the HSP90 inhibitor AUY922 (left panel) or HSP990 (right panel) for 24 h. Subsequently, the expression of pErk1/2, Erk1/2, pAkt, Akt, pp70S6K, p70S6K, p4EBP1 and 4EBP1 was evaluated by western blot analysis. A representative blot out of 3 independently performed experiments is shown.

    Journal: International Journal of Oncology

    Article Title: Potent antitumor activity of the novel HSP90 inhibitors AUY922 and HSP990 in neuroendocrine carcinoid cells

    doi: 10.3892/ijo.2013.2130

    Figure Lengend Snippet: Effect of HSP90 inhibition on downstream signaling. BON1, NCI-H727 and GOT1 cells were treated with increasing concentrations (1–100 nM) of the HSP90 inhibitor AUY922 (left panel) or HSP990 (right panel) for 24 h. Subsequently, the expression of pErk1/2, Erk1/2, pAkt, Akt, pp70S6K, p70S6K, p4EBP1 and 4EBP1 was evaluated by western blot analysis. A representative blot out of 3 independently performed experiments is shown.

    Article Snippet: After blocking in 2% non-fat dried milk, the membranes were incubated overnight in appropriate dilutions of antibodies against pAkt (Ser 473) (1:20,000), Akt (1:5,000), pErk 1/2 (1:10,000), Erk 1/2 (1:20,000), PARP (1:1,000), IGFR (1:5,000), p70S6K (1:1,000), pp70S6K (1:2,000), 4EBP1 (1:2,000) p4EBP1 (1:1,000) (all from Cell Signaling, Danvers, MA, USA), HSP70 (1:10,000) (Biomol Stressgen, Hamburg, Germany), HSP90 (1:5,000), EGFR (1:1,000), ErbB2 (1:500), ErbB3 (1:1,000) and STAT3 (1:10,000) (all from Santa Cruz, Heidelberg, Germany).

    Techniques: Inhibition, Expressing, Western Blot

    A, Odorant-induced Erk1/2 activation. Cultures were treated with vehicle, forskolin (FSK), 8-br-cAMP or odorants in the presence or absence of U0126. Odorants increased pErk1/2 levels, whereas forskolin and 8-br-cAMP did not. Preincubation with U0126

    Journal: Journal of neurochemistry

    Article Title: Phosphoinositide and Erk signaling pathways mediate activity-driven rodent olfactory sensory neuronal survival and stress mitigation

    doi: 10.1111/jnc.13131

    Figure Lengend Snippet: A, Odorant-induced Erk1/2 activation. Cultures were treated with vehicle, forskolin (FSK), 8-br-cAMP or odorants in the presence or absence of U0126. Odorants increased pErk1/2 levels, whereas forskolin and 8-br-cAMP did not. Preincubation with U0126

    Article Snippet: Anti-Erk1/2 and phospho-specific antibodies were commercially available (Erk1/2, #9102; phosphorylated Erk1/2, #9101; Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Activation Assay

    A, Odorants increased Erk2 phosphorylation in vivo . Mice (n=3) were exposed to a vehicle (1 ml of 2% EtOH) or odorant mixture (1 ml of 1 mM odorant mixtures in 2% EtOH: IBMP, citralva, and isovaleric acid) for 90 min in separate cages. Activation of Erk1/2

    Journal: Journal of neurochemistry

    Article Title: Phosphoinositide and Erk signaling pathways mediate activity-driven rodent olfactory sensory neuronal survival and stress mitigation

    doi: 10.1111/jnc.13131

    Figure Lengend Snippet: A, Odorants increased Erk2 phosphorylation in vivo . Mice (n=3) were exposed to a vehicle (1 ml of 2% EtOH) or odorant mixture (1 ml of 1 mM odorant mixtures in 2% EtOH: IBMP, citralva, and isovaleric acid) for 90 min in separate cages. Activation of Erk1/2

    Article Snippet: Anti-Erk1/2 and phospho-specific antibodies were commercially available (Erk1/2, #9102; phosphorylated Erk1/2, #9101; Cell Signaling Technology, Beverly, MA, USA).

    Techniques: In Vivo, Mouse Assay, Activation Assay