proteinase k treated torula yeast rna  (Millipore)


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    Structured Review

    Millipore proteinase k treated torula yeast rna
    Proteinase K Treated Torula Yeast Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81/100 stars

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    Clone Assay:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: Most of the multiple cloning site of the pBluescript vector between the RNA polymerase promoter and the insert had to be removed for the synthesis of riboprobes, because it generated an unacceptably high background signal (F.W., unpublished observation; ref. ). .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    Negative Control:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: Sense riboprobe was used as negative control. .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    Labeling:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: No labeling was observed with sense probes. .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    Concentration Assay:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: Digoxigenylated riboprobes were used for hybridization at a concentration of 3–5 μg/ml. .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    Generated:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: Most of the multiple cloning site of the pBluescript vector between the RNA polymerase promoter and the insert had to be removed for the synthesis of riboprobes, because it generated an unacceptably high background signal (F.W., unpublished observation; ref. ). .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    In Situ Hybridization:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: Paragraph title: In Situ Hybridization with Odorant Receptor Probes. ... Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    Plasmid Preparation:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: Most of the multiple cloning site of the pBluescript vector between the RNA polymerase promoter and the insert had to be removed for the synthesis of riboprobes, because it generated an unacceptably high background signal (F.W., unpublished observation; ref. ). .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml.

    Hybridization:

    Article Title: Nested expression domains for odorant receptors in zebrafish olfactory epithelium
    Article Snippet: .. Hybridization was performed overnight at 60°C in 50% formamide, 5× Denhardt’s reagent, 5× standard saline citrate (SSC; 20× SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 0.4 mg of proteinase K-treated torula yeast RNA per ml (Type VI, Sigma), and 0.1 mg of tRNA from bakers’ yeast per ml. ..

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    Millipore cathepsin l inhibitor iii
    Cathepsin usage by the HCoV-229E spike protein. (A) Effects of amino acid substitutions in the S protein on virus entry. Five amino acid substitutions (R642M, T681R, N714K, V765A, and A775S) are present around the fusion peptide sequence in the S protein of 229E/lab and 229E/clin-Sd. VSV-pseudotyped viruses bearing mutated S proteins or VSV-G were inoculated onto HeLa or HeLa-TMPRSS2 cells, and the GFP-positive cells were counted at 24 h postinfection ( n = 6). The data are expressed as percentages relative to those in HeLa-TMPRSS2 cells. (B) Effects of protease inhibitors on pseudotyped viruses bearing S proteins harboring R642M and N714K. HeLa or HeLa-TMPRSS2 cells were inoculated with VSV-pseudotyped viruses in the presence of inhibitors of <t>cathepsin</t> L (CatL) (cathepsin inhibitor <t>III)</t> or cathepsin B (CatB) (CA-074) (each at 10 μM). DMSO-treated cells served as a negative control. At least 200 GFP-positive cells/well were counted at 20 h postinfection ( n = 6). The data are expressed as percentages relative to those in HeLa cells treated with DMSO. n.s. (not significant), P > 0.05; * (significant), P ≤ 0.05; ** (highly significant), P ≤ 0.01. The error bars indicate SD.
    Cathepsin L Inhibitor Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 20s proteasome activity assay kit
    <t>Proteasome</t> activity does not contribute to the enhancement of degradation rates in quiescent cells. ( A ) Proteasome activity is enhanced in quiescent cells based on in vitro chymotrypsin-like activity assays; −epx (epoxomicin) indicates total chymotrypsin-like activity, +epx indicates chymotrypsin-like activity from nonproteasome sources, and the difference (∆) is a measurement of chymotrypsin-like activity from the proteasome. ( B ) Changes in the mRNA and protein expression levels of proteasome subunits determined by RNA-Seq and SILAC. The data indicate the up-regulation of 11S subunits PSME1 (PA28α) and PSME2 (PA28β) and were validated by ( C ) qPCR and ( D ) Western blots. The <t>20S</t> core subunit PSMB1 is included as a control. ( E ) Knockdown of PSME1 by shRNA. ( F ) Knockdown of PSME1 eliminates the enhancement of proteasome activity in quiescent cells based on chymotrypsin-like activity assays. ( G ) Knockdown of PSME1 does not influence protein degradation rates ( k degradation ) in quiescent cells as determined by dynamic SILAC experiments. ( H ) Expression level of a fluorescent proteasome substrate (pZsProSensor1) is unaffected by quiescence. Fibroblasts were transfected with pZsProSensor1 expression vector in dividing or contact-inhibited quiescent states, with or without subsequent addition of epoxomicin. The fluorescence distribution of cells was analyzed by FACS. Box plots were generated as described in Fig. 3 D . Comparisons were conducted by two-sided Mann−Whitney U test. NS (not significant) indicates a P value greater than 0.05.
    20s Proteasome Activity Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore proteasome activity
    Collagen type I does not affect prolyl hydroxylases activity, increases <t>proteasome</t> activity and proteasome inhibition restores erythropoietin levels. Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. ( A ) PHD 2 mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV as control. ( B ) PHD 2 protein levels and GAPDH as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( C ) Cells were grown on COLIV or COLI for 24 hrs and then treated with proteasome inhibitor MG 132 for another 24 hrs. Hydroxylated‐ HIF 1α protein levels and actin as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( D ) Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. Proteasome activity was measured as fluorogenic chymotrypsin substrate in a fluorimeter. Data are shown as the percentage of activity measured on COLIV . Cells were grown on COL IV or COLI for 24 hrs and then treated with proteasome inhibitor MG 132 for another 24 hrs. ( E ) Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . Data are shown as the percentage of activity measured on COLIV . ( F ) HIF 2α protein levels and GAPDH as endogenous control were evaluated by immunoblotting. A representative blot is shown. Bar graphs represent percentage of densitometric levels versus COLIV as control. Results are mean ± S.E.M.; N = 3 independent experiments (* P
    Proteasome Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cathepsin usage by the HCoV-229E spike protein. (A) Effects of amino acid substitutions in the S protein on virus entry. Five amino acid substitutions (R642M, T681R, N714K, V765A, and A775S) are present around the fusion peptide sequence in the S protein of 229E/lab and 229E/clin-Sd. VSV-pseudotyped viruses bearing mutated S proteins or VSV-G were inoculated onto HeLa or HeLa-TMPRSS2 cells, and the GFP-positive cells were counted at 24 h postinfection ( n = 6). The data are expressed as percentages relative to those in HeLa-TMPRSS2 cells. (B) Effects of protease inhibitors on pseudotyped viruses bearing S proteins harboring R642M and N714K. HeLa or HeLa-TMPRSS2 cells were inoculated with VSV-pseudotyped viruses in the presence of inhibitors of cathepsin L (CatL) (cathepsin inhibitor III) or cathepsin B (CatB) (CA-074) (each at 10 μM). DMSO-treated cells served as a negative control. At least 200 GFP-positive cells/well were counted at 20 h postinfection ( n = 6). The data are expressed as percentages relative to those in HeLa cells treated with DMSO. n.s. (not significant), P > 0.05; * (significant), P ≤ 0.05; ** (highly significant), P ≤ 0.01. The error bars indicate SD.

    Journal: Journal of Virology

    Article Title: Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry

    doi: 10.1128/JVI.01387-16

    Figure Lengend Snippet: Cathepsin usage by the HCoV-229E spike protein. (A) Effects of amino acid substitutions in the S protein on virus entry. Five amino acid substitutions (R642M, T681R, N714K, V765A, and A775S) are present around the fusion peptide sequence in the S protein of 229E/lab and 229E/clin-Sd. VSV-pseudotyped viruses bearing mutated S proteins or VSV-G were inoculated onto HeLa or HeLa-TMPRSS2 cells, and the GFP-positive cells were counted at 24 h postinfection ( n = 6). The data are expressed as percentages relative to those in HeLa-TMPRSS2 cells. (B) Effects of protease inhibitors on pseudotyped viruses bearing S proteins harboring R642M and N714K. HeLa or HeLa-TMPRSS2 cells were inoculated with VSV-pseudotyped viruses in the presence of inhibitors of cathepsin L (CatL) (cathepsin inhibitor III) or cathepsin B (CatB) (CA-074) (each at 10 μM). DMSO-treated cells served as a negative control. At least 200 GFP-positive cells/well were counted at 20 h postinfection ( n = 6). The data are expressed as percentages relative to those in HeLa cells treated with DMSO. n.s. (not significant), P > 0.05; * (significant), P ≤ 0.05; ** (highly significant), P ≤ 0.01. The error bars indicate SD.

    Article Snippet: The following inhibitors were used: E64d (330005; Calbiochem, San Diego, CA, USA), camostat mesylate (3193; Tocris Bioscience, Bristol, UK), cathepsin L inhibitor III (219427; Calbiochem), and the cathepsin B inhibitor CA-074 (C5732; Sigma).

    Techniques: Sequencing, Negative Control

    Replication of the passaged HCoV-229E clinical isolate in HBTE-ALI cells. (A) Characterization of HBTE cells by HCoV-HKU-1 infection. HBTE cells were cultured for 4 weeks in differentiation medium at an air-liquid interface (HBTE-ALI) in a 6.5-mm-diameter Transwell chamber. To confirm differentiation, HBTE cells, differentiated (incubated for 4 weeks) or undifferentiated (incubated for 0 weeks), were inoculated with HCoV-HKU1 ( n = 2). After 2 h, the inoculated virus was removed and the cells were washed three times with medium. After 72 h of incubation at the ALI, RNA was collected from the medium, and real-time PCR was performed to quantify viral RNA. (B) Characterization of HBTE cells by measurement of cellular transcripts. Expression of cellular mRNAs encoding cell differentiation markers (E-cadherin, ZO-1, and MUC5AC) and factors associated with HCoV-229E infection (APN, TMPRSS2, and cathepsin L) in differentiated and undifferentiated HBTE cells was measured by real-time PCR ( n = 4). The data are expressed as the fold change in transcript levels in differentiated cells relative to that in undifferentiated cells. (C) Replication of passaged HCoV-229E in HBTE cells. HBTE-ALI cells were inoculated with 229E/clin-Sd-p1 and 229E/clin-Sd-p20 (10 4 PFU), the titers of which were measured in HeLa-TMPRSS2 cells cultured in medium supplemented with trypsin ( n = 4). HeLa cells were used as a control to confirm differential RNA expression due to the differing abilities of passage 1 and passage 20 virus to use cathepsin. After 1 h, the inoculated virus was removed, and the cells were washed three times in culture medium. After 24 h of incubation at the ALI, cellular RNA was collected, and viral RNA was measured by real-time PCR. ** (highly significant), P ≤ 0.01; *** (very highly significant), P ≤ 0.001. The error bars indicate SD.

    Journal: Journal of Virology

    Article Title: Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry

    doi: 10.1128/JVI.01387-16

    Figure Lengend Snippet: Replication of the passaged HCoV-229E clinical isolate in HBTE-ALI cells. (A) Characterization of HBTE cells by HCoV-HKU-1 infection. HBTE cells were cultured for 4 weeks in differentiation medium at an air-liquid interface (HBTE-ALI) in a 6.5-mm-diameter Transwell chamber. To confirm differentiation, HBTE cells, differentiated (incubated for 4 weeks) or undifferentiated (incubated for 0 weeks), were inoculated with HCoV-HKU1 ( n = 2). After 2 h, the inoculated virus was removed and the cells were washed three times with medium. After 72 h of incubation at the ALI, RNA was collected from the medium, and real-time PCR was performed to quantify viral RNA. (B) Characterization of HBTE cells by measurement of cellular transcripts. Expression of cellular mRNAs encoding cell differentiation markers (E-cadherin, ZO-1, and MUC5AC) and factors associated with HCoV-229E infection (APN, TMPRSS2, and cathepsin L) in differentiated and undifferentiated HBTE cells was measured by real-time PCR ( n = 4). The data are expressed as the fold change in transcript levels in differentiated cells relative to that in undifferentiated cells. (C) Replication of passaged HCoV-229E in HBTE cells. HBTE-ALI cells were inoculated with 229E/clin-Sd-p1 and 229E/clin-Sd-p20 (10 4 PFU), the titers of which were measured in HeLa-TMPRSS2 cells cultured in medium supplemented with trypsin ( n = 4). HeLa cells were used as a control to confirm differential RNA expression due to the differing abilities of passage 1 and passage 20 virus to use cathepsin. After 1 h, the inoculated virus was removed, and the cells were washed three times in culture medium. After 24 h of incubation at the ALI, cellular RNA was collected, and viral RNA was measured by real-time PCR. ** (highly significant), P ≤ 0.01; *** (very highly significant), P ≤ 0.001. The error bars indicate SD.

    Article Snippet: The following inhibitors were used: E64d (330005; Calbiochem, San Diego, CA, USA), camostat mesylate (3193; Tocris Bioscience, Bristol, UK), cathepsin L inhibitor III (219427; Calbiochem), and the cathepsin B inhibitor CA-074 (C5732; Sigma).

    Techniques: Infection, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Expressing, Cell Differentiation, RNA Expression

    Proteasome activity does not contribute to the enhancement of degradation rates in quiescent cells. ( A ) Proteasome activity is enhanced in quiescent cells based on in vitro chymotrypsin-like activity assays; −epx (epoxomicin) indicates total chymotrypsin-like activity, +epx indicates chymotrypsin-like activity from nonproteasome sources, and the difference (∆) is a measurement of chymotrypsin-like activity from the proteasome. ( B ) Changes in the mRNA and protein expression levels of proteasome subunits determined by RNA-Seq and SILAC. The data indicate the up-regulation of 11S subunits PSME1 (PA28α) and PSME2 (PA28β) and were validated by ( C ) qPCR and ( D ) Western blots. The 20S core subunit PSMB1 is included as a control. ( E ) Knockdown of PSME1 by shRNA. ( F ) Knockdown of PSME1 eliminates the enhancement of proteasome activity in quiescent cells based on chymotrypsin-like activity assays. ( G ) Knockdown of PSME1 does not influence protein degradation rates ( k degradation ) in quiescent cells as determined by dynamic SILAC experiments. ( H ) Expression level of a fluorescent proteasome substrate (pZsProSensor1) is unaffected by quiescence. Fibroblasts were transfected with pZsProSensor1 expression vector in dividing or contact-inhibited quiescent states, with or without subsequent addition of epoxomicin. The fluorescence distribution of cells was analyzed by FACS. Box plots were generated as described in Fig. 3 D . Comparisons were conducted by two-sided Mann−Whitney U test. NS (not significant) indicates a P value greater than 0.05.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Proteome-wide modulation of degradation dynamics in response to growth arrest

    doi: 10.1073/pnas.1710238114

    Figure Lengend Snippet: Proteasome activity does not contribute to the enhancement of degradation rates in quiescent cells. ( A ) Proteasome activity is enhanced in quiescent cells based on in vitro chymotrypsin-like activity assays; −epx (epoxomicin) indicates total chymotrypsin-like activity, +epx indicates chymotrypsin-like activity from nonproteasome sources, and the difference (∆) is a measurement of chymotrypsin-like activity from the proteasome. ( B ) Changes in the mRNA and protein expression levels of proteasome subunits determined by RNA-Seq and SILAC. The data indicate the up-regulation of 11S subunits PSME1 (PA28α) and PSME2 (PA28β) and were validated by ( C ) qPCR and ( D ) Western blots. The 20S core subunit PSMB1 is included as a control. ( E ) Knockdown of PSME1 by shRNA. ( F ) Knockdown of PSME1 eliminates the enhancement of proteasome activity in quiescent cells based on chymotrypsin-like activity assays. ( G ) Knockdown of PSME1 does not influence protein degradation rates ( k degradation ) in quiescent cells as determined by dynamic SILAC experiments. ( H ) Expression level of a fluorescent proteasome substrate (pZsProSensor1) is unaffected by quiescence. Fibroblasts were transfected with pZsProSensor1 expression vector in dividing or contact-inhibited quiescent states, with or without subsequent addition of epoxomicin. The fluorescence distribution of cells was analyzed by FACS. Box plots were generated as described in Fig. 3 D . Comparisons were conducted by two-sided Mann−Whitney U test. NS (not significant) indicates a P value greater than 0.05.

    Article Snippet: In vitro proteasome activity was measured by using 20S Proteasome activity assay kit (Millipore); 75 μg of total protein in 50 μL of cell lysate was incubated in assay buffer containing 25 nM Hepes, pH 7.4, 0.05 mL of EDTA, 0.05% mM Nonidet P-40, 0.001% SDS, and 25 μg of the proteasome substrate, LLVY-AMC, for 90 min at 37 °C.

    Techniques: Activity Assay, In Vitro, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, shRNA, Transfection, Plasmid Preparation, Fluorescence, FACS, Generated, MANN-WHITNEY

    Collagen type I does not affect prolyl hydroxylases activity, increases proteasome activity and proteasome inhibition restores erythropoietin levels. Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. ( A ) PHD 2 mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV as control. ( B ) PHD 2 protein levels and GAPDH as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( C ) Cells were grown on COLIV or COLI for 24 hrs and then treated with proteasome inhibitor MG 132 for another 24 hrs. Hydroxylated‐ HIF 1α protein levels and actin as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( D ) Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. Proteasome activity was measured as fluorogenic chymotrypsin substrate in a fluorimeter. Data are shown as the percentage of activity measured on COLIV . Cells were grown on COL IV or COLI for 24 hrs and then treated with proteasome inhibitor MG 132 for another 24 hrs. ( E ) Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . Data are shown as the percentage of activity measured on COLIV . ( F ) HIF 2α protein levels and GAPDH as endogenous control were evaluated by immunoblotting. A representative blot is shown. Bar graphs represent percentage of densitometric levels versus COLIV as control. Results are mean ± S.E.M.; N = 3 independent experiments (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition

    doi: 10.1111/jcmm.13319

    Figure Lengend Snippet: Collagen type I does not affect prolyl hydroxylases activity, increases proteasome activity and proteasome inhibition restores erythropoietin levels. Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. ( A ) PHD 2 mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV as control. ( B ) PHD 2 protein levels and GAPDH as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( C ) Cells were grown on COLIV or COLI for 24 hrs and then treated with proteasome inhibitor MG 132 for another 24 hrs. Hydroxylated‐ HIF 1α protein levels and actin as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( D ) Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. Proteasome activity was measured as fluorogenic chymotrypsin substrate in a fluorimeter. Data are shown as the percentage of activity measured on COLIV . Cells were grown on COL IV or COLI for 24 hrs and then treated with proteasome inhibitor MG 132 for another 24 hrs. ( E ) Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . Data are shown as the percentage of activity measured on COLIV . ( F ) HIF 2α protein levels and GAPDH as endogenous control were evaluated by immunoblotting. A representative blot is shown. Bar graphs represent percentage of densitometric levels versus COLIV as control. Results are mean ± S.E.M.; N = 3 independent experiments (* P

    Article Snippet: FAK inhibitor 4‐amino‐5‐(4‐chloro‐phenyl)‐7‐(t ‐butyl)pyrazolo3,4‐dpyrimidine (PP2), Fluorogenic substrate Succ‐LLVY‐AMC for proteasome activity and proteasome inhibitor MG132 were purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Activity Assay, Inhibition, Quantitative RT-PCR

    Deleted FAK restored proteasome activity at basal levels, inhibits HIF ‐2α degradation and restores erythropoietin levels. Cells were depleted of FAK with a specific small‐interfering RNA (si FAK ), and a scrambled RNA (Scramble) was used as control. ( A ) Expression of Total‐ FAK protein was analysed by Western blot. Equal protein loading was confirmed by probing with actin as endogenous control. A representative Western blot is shown. Cells transfected with si RNA against FAK protein or scramble were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. Then, it was determined: ( B ) Proteasome activity measured as fluorogenic chymotrypsin substrate in a fluorimeter. Data are shown as the percentage of activity measured on COLIV . ( C ) HIF 2α protein levels and actin as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( D ) Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . ( E ) Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 24 hrs and then incubated with the FAK inhibitor 4‐amino‐5‐(4‐chloro‐phenyl)‐7‐( t ‐butyl) pyrazolo3,4‐dpyrimidine ( PP 2) 10 μM for other 24 hrs. Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . Results are mean ± S.E.M.; N = 3 independent experiments (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition

    doi: 10.1111/jcmm.13319

    Figure Lengend Snippet: Deleted FAK restored proteasome activity at basal levels, inhibits HIF ‐2α degradation and restores erythropoietin levels. Cells were depleted of FAK with a specific small‐interfering RNA (si FAK ), and a scrambled RNA (Scramble) was used as control. ( A ) Expression of Total‐ FAK protein was analysed by Western blot. Equal protein loading was confirmed by probing with actin as endogenous control. A representative Western blot is shown. Cells transfected with si RNA against FAK protein or scramble were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 48 hrs. Then, it was determined: ( B ) Proteasome activity measured as fluorogenic chymotrypsin substrate in a fluorimeter. Data are shown as the percentage of activity measured on COLIV . ( C ) HIF 2α protein levels and actin as endogenous control were evaluated by immunoblotting. A representative blot is shown. ( D ) Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . ( E ) Cells were grown on collagen IV ( COLIV ) or collagen I ( COLI ) for 24 hrs and then incubated with the FAK inhibitor 4‐amino‐5‐(4‐chloro‐phenyl)‐7‐( t ‐butyl) pyrazolo3,4‐dpyrimidine ( PP 2) 10 μM for other 24 hrs. Erythropoietin mRNA levels were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold induction over COLIV . Results are mean ± S.E.M.; N = 3 independent experiments (* P

    Article Snippet: FAK inhibitor 4‐amino‐5‐(4‐chloro‐phenyl)‐7‐(t ‐butyl)pyrazolo3,4‐dpyrimidine (PP2), Fluorogenic substrate Succ‐LLVY‐AMC for proteasome activity and proteasome inhibitor MG132 were purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Activity Assay, Small Interfering RNA, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Incubation

    EPO mRNA is recovered in obstructed kidneys of mice with UUO treated with proteasome inhibitor bortezomib. ( A ) Representative images of the renal cortex stained with ( A ) Masson's trichrome and ( B ) Sirius red and quantification by Image‐Pro Plus expressed as square microns corresponding to non‐obstructed ( NO ), obstructed (O), non‐obstructed treated with bortezomib ( NO ‐B) or obstructed treated with bortezomib (O‐B) kidneys. Bar = 100 μm. ( C ) Erythropoietin mRNA levels from renal cortex from NO , O, NO ‐B and O‐B kidneys were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold changes over non‐obstructed ( NO ) kidney. Data are means ± S.E. of five to seven animals/group. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition

    doi: 10.1111/jcmm.13319

    Figure Lengend Snippet: EPO mRNA is recovered in obstructed kidneys of mice with UUO treated with proteasome inhibitor bortezomib. ( A ) Representative images of the renal cortex stained with ( A ) Masson's trichrome and ( B ) Sirius red and quantification by Image‐Pro Plus expressed as square microns corresponding to non‐obstructed ( NO ), obstructed (O), non‐obstructed treated with bortezomib ( NO ‐B) or obstructed treated with bortezomib (O‐B) kidneys. Bar = 100 μm. ( C ) Erythropoietin mRNA levels from renal cortex from NO , O, NO ‐B and O‐B kidneys were determined by quantitative RT ‐ PCR . Relative fold change values were normalized against GAPDH as endogenous control and expressed as fold changes over non‐obstructed ( NO ) kidney. Data are means ± S.E. of five to seven animals/group. * P

    Article Snippet: FAK inhibitor 4‐amino‐5‐(4‐chloro‐phenyl)‐7‐(t ‐butyl)pyrazolo3,4‐dpyrimidine (PP2), Fluorogenic substrate Succ‐LLVY‐AMC for proteasome activity and proteasome inhibitor MG132 were purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Mouse Assay, Staining, Quantitative RT-PCR