anti total 4e bp1  (Santa Cruz Biotechnology)

 
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    Santa Cruz Biotechnology anti total 4e bp1
    EDL muscles were held at optimal length ( Lo ) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, <t>and</t> <t>4E-BP1.</t> The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
    Anti Total 4e Bp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mechanical Stimulation Induces mTOR Signaling via an ERK-Independent Mechanism: Implications for a Direct Activation of mTOR by Phosphatidic Acid"

    Article Title: Mechanical Stimulation Induces mTOR Signaling via an ERK-Independent Mechanism: Implications for a Direct Activation of mTOR by Phosphatidic Acid

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047258

    EDL muscles were held at optimal length ( Lo ) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
    Figure Legend Snippet: EDL muscles were held at optimal length ( Lo ) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Techniques Used: Ex Vivo, Organ Culture, Incubation, Western Blot

    ( A ) EDL muscles from wild type mice, and ( B ) transgenic mice with muscle specific expression of a rapamycin-resistant mutant of mTOR (RR-mTOR), were held at Lo and pre-incubated with 150 nM rapamycin (RAP +) or the vehicle (RAP –, DMSO) for 30 min. The muscles were then subjected to 90 min of stretch or control conditions followed by western blot analysis for phosphorylated (P) and total p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the genotype-matched vehicle control samples (RAP–, Stretch–). Note: total 4E-BP1 gels were also run under conditions that minimize the gel mobility shift (short-run). All values are presented as the mean (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
    Figure Legend Snippet: ( A ) EDL muscles from wild type mice, and ( B ) transgenic mice with muscle specific expression of a rapamycin-resistant mutant of mTOR (RR-mTOR), were held at Lo and pre-incubated with 150 nM rapamycin (RAP +) or the vehicle (RAP –, DMSO) for 30 min. The muscles were then subjected to 90 min of stretch or control conditions followed by western blot analysis for phosphorylated (P) and total p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the genotype-matched vehicle control samples (RAP–, Stretch–). Note: total 4E-BP1 gels were also run under conditions that minimize the gel mobility shift (short-run). All values are presented as the mean (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Techniques Used: Transgenic Assay, Expressing, Mutagenesis, Incubation, Western Blot, Mobility Shift

    ( A ) EDL muscles were held at Lo and pre-labeled with [ 3 H]-myristic acid for 2 h. The muscles were then subjected to 15 or 90 min of stretch or control conditions. Muscles were collected at the end of the 15 or 90 min interval, and the amount of 3 H-labeled PA ( 3 H PA) was measured and then expressed as a percentage of time-matched control values. The values are presented as the mean + SEM (n = 4−7 per group). • Significantly different from the time-matched control group. ( B ) C2C12 myoblasts were serum-starved overnight and then pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min, followed by 20 min stimulation with 30 µM C8 PA, 300 µM Egg PA or the vehicle (Control, PBS). The samples were then subjected to western blot analysis for phosphorylated (P) and total ERK, p70, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the vehicle control samples (U0126 –, PBS). All values are presented as the mean and were obtained from three independent experiments (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
    Figure Legend Snippet: ( A ) EDL muscles were held at Lo and pre-labeled with [ 3 H]-myristic acid for 2 h. The muscles were then subjected to 15 or 90 min of stretch or control conditions. Muscles were collected at the end of the 15 or 90 min interval, and the amount of 3 H-labeled PA ( 3 H PA) was measured and then expressed as a percentage of time-matched control values. The values are presented as the mean + SEM (n = 4−7 per group). • Significantly different from the time-matched control group. ( B ) C2C12 myoblasts were serum-starved overnight and then pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min, followed by 20 min stimulation with 30 µM C8 PA, 300 µM Egg PA or the vehicle (Control, PBS). The samples were then subjected to western blot analysis for phosphorylated (P) and total ERK, p70, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the vehicle control samples (U0126 –, PBS). All values are presented as the mean and were obtained from three independent experiments (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Techniques Used: Labeling, Incubation, Western Blot

    antibodies against total 4e bp1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology antibodies against total 4e bp1
    (A) Wild-type and DKO MEFs were pre-incubated in complete medium in the presence of additional NaCl at the concentrations indicated. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and total protein synthesis was then measured by the incorporation of [ 35 S]methionine (2 µCi/ml) for 1 h as described in Experimental. The data are the means of triplicate determinations and are expressed as counts per min incorporated per µg of protein ± S.E.M. Significances of differences between incubations ± Ku-0063794 were determined by unpaired t tests: * = p<0.005; N.S. = not significant. (B) Extracts from <t>4E-BP</t> wild-type and DKO cells were analysed for expression <t>of</t> <t>4E-BP1</t> by immunoblotting. Blots for GAPDH are also shown as loading controls. (C) Summary of % inhibition of protein synthesis by Ku-0063794 in 4E-BP wild-type (wt) and DKO cells as a function of the additional NaCl concentration.
    Antibodies Against Total 4e Bp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition"

    Article Title: Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071138

    (A) Wild-type and DKO MEFs were pre-incubated in complete medium in the presence of additional NaCl at the concentrations indicated. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and total protein synthesis was then measured by the incorporation of [ 35 S]methionine (2 µCi/ml) for 1 h as described in Experimental. The data are the means of triplicate determinations and are expressed as counts per min incorporated per µg of protein ± S.E.M. Significances of differences between incubations ± Ku-0063794 were determined by unpaired t tests: * = p<0.005; N.S. = not significant. (B) Extracts from 4E-BP wild-type and DKO cells were analysed for expression of 4E-BP1 by immunoblotting. Blots for GAPDH are also shown as loading controls. (C) Summary of % inhibition of protein synthesis by Ku-0063794 in 4E-BP wild-type (wt) and DKO cells as a function of the additional NaCl concentration.
    Figure Legend Snippet: (A) Wild-type and DKO MEFs were pre-incubated in complete medium in the presence of additional NaCl at the concentrations indicated. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and total protein synthesis was then measured by the incorporation of [ 35 S]methionine (2 µCi/ml) for 1 h as described in Experimental. The data are the means of triplicate determinations and are expressed as counts per min incorporated per µg of protein ± S.E.M. Significances of differences between incubations ± Ku-0063794 were determined by unpaired t tests: * = p<0.005; N.S. = not significant. (B) Extracts from 4E-BP wild-type and DKO cells were analysed for expression of 4E-BP1 by immunoblotting. Blots for GAPDH are also shown as loading controls. (C) Summary of % inhibition of protein synthesis by Ku-0063794 in 4E-BP wild-type (wt) and DKO cells as a function of the additional NaCl concentration.

    Techniques Used: Incubation, Expressing, Western Blot, Inhibition, Concentration Assay

    4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. The extracts were analysed for total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ), total and phosphorylated Akt (Ser 473 ), and total and phosphorylated p70S6kinase (Thr 421 /Ser 424 and Thr 389 ) by SDS gel electrophoresis and immunoblotting. The positions of the differentially phosphorylated α, β and γ forms and of a cleavage product of 4E-BP1 are indicated. Blots for GAPDH are also shown as loading controls.
    Figure Legend Snippet: 4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. The extracts were analysed for total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ), total and phosphorylated Akt (Ser 473 ), and total and phosphorylated p70S6kinase (Thr 421 /Ser 424 and Thr 389 ) by SDS gel electrophoresis and immunoblotting. The positions of the differentially phosphorylated α, β and γ forms and of a cleavage product of 4E-BP1 are indicated. Blots for GAPDH are also shown as loading controls.

    Techniques Used: Incubation, SDS-Gel, Electrophoresis, Western Blot

    4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. Using equal quantities of total protein, the extracts were then subjected to affinity chromatography on m 7 GTP-Sepharose to isolate eIF4E and its associated proteins, as described in Experimental. The bound proteins were analysed for eIF4E, 4E-BP1 and eIF4GI by SDS gel electrophoresis and immunoblotting.
    Figure Legend Snippet: 4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. Using equal quantities of total protein, the extracts were then subjected to affinity chromatography on m 7 GTP-Sepharose to isolate eIF4E and its associated proteins, as described in Experimental. The bound proteins were analysed for eIF4E, 4E-BP1 and eIF4GI by SDS gel electrophoresis and immunoblotting.

    Techniques Used: Incubation, Affinity Chromatography, SDS-Gel, Electrophoresis, Western Blot

    total 4e bp1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology total 4e bp1
    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total <t>p70S6K,</t> <t>p-4E-BP1</t> (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.
    Total 4e Bp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway"

    Article Title: Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103071

    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.
    Figure Legend Snippet: HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.

    Techniques Used: Incubation, Injection, Western Blot

    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (A) and livers (B). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. A polysomal analysis was performed to detect CD36 translational efficiency in the cells (C) and livers (D). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at P <0.05, and # denotes a significant difference at P <0.01.
    Figure Legend Snippet: HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (A) and livers (B). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. A polysomal analysis was performed to detect CD36 translational efficiency in the cells (C) and livers (D). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at P <0.05, and # denotes a significant difference at P <0.01.

    Techniques Used: Incubation, Injection, Western Blot

    total 4ebp1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology total 4ebp1
    Total 4ebp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    total 4e bp1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology total 4e bp1
    ( A ) Uridine incorporation in the CLL lymphocytes. Cells were treated with dimethyl sulfoxide (DMSO) or indicated concentrations of AZD1208 for 23 h and then incubated with radioactive uridine for 1 h followed by radioactivity measured via scintillation counter. Data were normalized to cell number and were plotted as the percentage of untreated control. Treatments for each CLL sample were done in triplicate, and the bars represent the mean ± SEM. ( B ) Leucine incorporation in CLL lymphocytes. Cells were treated with DMSO or indicated concentrations of AZD1208 for 23 h and then incubated with radioactive leucine for 1 h followed by radioactivity measured via scintillation counter. Data were normalized to cell number and were plotted as the percentage of untreated control. Treatments for each CLL sample were done in triplicate, and the bars represent the mean ± SEM. ( C ) Effect of AZD1208 on phospho-c-MYC (Ser62), total c-MYC, phospho-histone H3 (Ser10), and total histone H3. CLL cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24 h; cells were then harvested, lysed, and analyzed via immunoblot. Phosphorylated-to-total protein ratios were calculated, and the numbers are depicted below the phosphorylated protein bands. ( D ) Effect of AZD1208 on phospho-p70S6K <t>(Thr389),</t> <t>phospho-4E-BP1</t> (Ser65), phospho-4E-BP1 (Thr37/46), and total 4E-BP1 protein. Cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24 h; cells were then harvested, lysed, and analyzed via immunoblot. ( E ) Effect of AZD1208 treatment for 24, 48, and 72 h on phospho-4E-BP1 Thr37/46 and total 4E-BP1. Cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24, 48, and 72 h; cells were then harvested, lysed, and analyzed via immunoblot. Phosphorylated-to-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein ratios were calculated, and the numbers are depicted below the phosphorylated protein bands. The membrane was probed with LI-COR imaging system using anti-rabbit and anti-mouse with distinct fluorescent wavelengths. When more than two proteins with the same molecular weight needed to be detected, the membrane was gently stripped and re-probed. In such cases, loading control remains same. Note: GAPDH loading control for Figure (bottom gel) and is the same. Note: GAPDH loading control in Figures and is the same.
    Total 4e Bp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PIM kinase inhibitor, AZD1208, inhibits protein translation and induces autophagy in primary chronic lymphocytic leukemia cells"

    Article Title: PIM kinase inhibitor, AZD1208, inhibits protein translation and induces autophagy in primary chronic lymphocytic leukemia cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26876

    ( A ) Uridine incorporation in the CLL lymphocytes. Cells were treated with dimethyl sulfoxide (DMSO) or indicated concentrations of AZD1208 for 23 h and then incubated with radioactive uridine for 1 h followed by radioactivity measured via scintillation counter. Data were normalized to cell number and were plotted as the percentage of untreated control. Treatments for each CLL sample were done in triplicate, and the bars represent the mean ± SEM. ( B ) Leucine incorporation in CLL lymphocytes. Cells were treated with DMSO or indicated concentrations of AZD1208 for 23 h and then incubated with radioactive leucine for 1 h followed by radioactivity measured via scintillation counter. Data were normalized to cell number and were plotted as the percentage of untreated control. Treatments for each CLL sample were done in triplicate, and the bars represent the mean ± SEM. ( C ) Effect of AZD1208 on phospho-c-MYC (Ser62), total c-MYC, phospho-histone H3 (Ser10), and total histone H3. CLL cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24 h; cells were then harvested, lysed, and analyzed via immunoblot. Phosphorylated-to-total protein ratios were calculated, and the numbers are depicted below the phosphorylated protein bands. ( D ) Effect of AZD1208 on phospho-p70S6K (Thr389), phospho-4E-BP1 (Ser65), phospho-4E-BP1 (Thr37/46), and total 4E-BP1 protein. Cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24 h; cells were then harvested, lysed, and analyzed via immunoblot. ( E ) Effect of AZD1208 treatment for 24, 48, and 72 h on phospho-4E-BP1 Thr37/46 and total 4E-BP1. Cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24, 48, and 72 h; cells were then harvested, lysed, and analyzed via immunoblot. Phosphorylated-to-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein ratios were calculated, and the numbers are depicted below the phosphorylated protein bands. The membrane was probed with LI-COR imaging system using anti-rabbit and anti-mouse with distinct fluorescent wavelengths. When more than two proteins with the same molecular weight needed to be detected, the membrane was gently stripped and re-probed. In such cases, loading control remains same. Note: GAPDH loading control for Figure (bottom gel) and is the same. Note: GAPDH loading control in Figures and is the same.
    Figure Legend Snippet: ( A ) Uridine incorporation in the CLL lymphocytes. Cells were treated with dimethyl sulfoxide (DMSO) or indicated concentrations of AZD1208 for 23 h and then incubated with radioactive uridine for 1 h followed by radioactivity measured via scintillation counter. Data were normalized to cell number and were plotted as the percentage of untreated control. Treatments for each CLL sample were done in triplicate, and the bars represent the mean ± SEM. ( B ) Leucine incorporation in CLL lymphocytes. Cells were treated with DMSO or indicated concentrations of AZD1208 for 23 h and then incubated with radioactive leucine for 1 h followed by radioactivity measured via scintillation counter. Data were normalized to cell number and were plotted as the percentage of untreated control. Treatments for each CLL sample were done in triplicate, and the bars represent the mean ± SEM. ( C ) Effect of AZD1208 on phospho-c-MYC (Ser62), total c-MYC, phospho-histone H3 (Ser10), and total histone H3. CLL cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24 h; cells were then harvested, lysed, and analyzed via immunoblot. Phosphorylated-to-total protein ratios were calculated, and the numbers are depicted below the phosphorylated protein bands. ( D ) Effect of AZD1208 on phospho-p70S6K (Thr389), phospho-4E-BP1 (Ser65), phospho-4E-BP1 (Thr37/46), and total 4E-BP1 protein. Cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24 h; cells were then harvested, lysed, and analyzed via immunoblot. ( E ) Effect of AZD1208 treatment for 24, 48, and 72 h on phospho-4E-BP1 Thr37/46 and total 4E-BP1. Cells were treated with the vehicle DMSO or AZD1208 (3 μM or 10 μM) for 24, 48, and 72 h; cells were then harvested, lysed, and analyzed via immunoblot. Phosphorylated-to-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein ratios were calculated, and the numbers are depicted below the phosphorylated protein bands. The membrane was probed with LI-COR imaging system using anti-rabbit and anti-mouse with distinct fluorescent wavelengths. When more than two proteins with the same molecular weight needed to be detected, the membrane was gently stripped and re-probed. In such cases, loading control remains same. Note: GAPDH loading control for Figure (bottom gel) and is the same. Note: GAPDH loading control in Figures and is the same.

    Techniques Used: Incubation, Radioactivity, Western Blot, Imaging, Molecular Weight

    total 4e bp1  (Santa Cruz Biotechnology)

     
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    A. Western blot analysis assessing the activation of signaling cascades in H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901), 2000 nM binimetinib (Bini), 100 nM trametinib (Tram) or 1000 nM ulixertinib (Ulix) for 24 hours. B. Western blot analysis assessing the activity of mTOR, S6 ribosomal <t>protein,</t> <t>4E-BP1</t> and TSC2 in parental H358 and H358-PR250 cells ( left panel ) or H460 and H460-PR500 cells ( right panel ). C. Colony formation assay assessing the sensitivity of parental H358 and H358-PR250 cells to treatment for 14 days with the indicated doses of everolimus ( left panel ) and MK2206 ( right panel ). D. Assessment of CDK and G1 cyclin expression by Western blot analysis in parental H358 and H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901), 2000 nM MK2206 (2206) or 100 nM everolimus (Eve) for 24 hours. E. Kinase activity assay examining the activity of CDK2 and CDK4 in parental H358 and H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901) or 100 nM everolimus (Eve) for 24 hours.
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    1) Product Images from "Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS -mutant non-small cell lung cancer"

    Article Title: Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS -mutant non-small cell lung cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25803

    A. Western blot analysis assessing the activation of signaling cascades in H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901), 2000 nM binimetinib (Bini), 100 nM trametinib (Tram) or 1000 nM ulixertinib (Ulix) for 24 hours. B. Western blot analysis assessing the activity of mTOR, S6 ribosomal protein, 4E-BP1 and TSC2 in parental H358 and H358-PR250 cells ( left panel ) or H460 and H460-PR500 cells ( right panel ). C. Colony formation assay assessing the sensitivity of parental H358 and H358-PR250 cells to treatment for 14 days with the indicated doses of everolimus ( left panel ) and MK2206 ( right panel ). D. Assessment of CDK and G1 cyclin expression by Western blot analysis in parental H358 and H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901), 2000 nM MK2206 (2206) or 100 nM everolimus (Eve) for 24 hours. E. Kinase activity assay examining the activity of CDK2 and CDK4 in parental H358 and H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901) or 100 nM everolimus (Eve) for 24 hours.
    Figure Legend Snippet: A. Western blot analysis assessing the activation of signaling cascades in H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901), 2000 nM binimetinib (Bini), 100 nM trametinib (Tram) or 1000 nM ulixertinib (Ulix) for 24 hours. B. Western blot analysis assessing the activity of mTOR, S6 ribosomal protein, 4E-BP1 and TSC2 in parental H358 and H358-PR250 cells ( left panel ) or H460 and H460-PR500 cells ( right panel ). C. Colony formation assay assessing the sensitivity of parental H358 and H358-PR250 cells to treatment for 14 days with the indicated doses of everolimus ( left panel ) and MK2206 ( right panel ). D. Assessment of CDK and G1 cyclin expression by Western blot analysis in parental H358 and H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901), 2000 nM MK2206 (2206) or 100 nM everolimus (Eve) for 24 hours. E. Kinase activity assay examining the activity of CDK2 and CDK4 in parental H358 and H358-PR250 cells treated with DMSO (D), 100 nM PD0325901 (901) or 100 nM everolimus (Eve) for 24 hours.

    Techniques Used: Western Blot, Activation Assay, Activity Assay, Colony Assay, Expressing, Kinase Assay

    4e bp1 total  (Santa Cruz Biotechnology)

     
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    Phosphorylation and total protein involved in protein synthesis in the gastrocnemius and soleus of bGH ( n = 9) and WT ( n = 9) mice. mTOR phosphorylation and mTOR total protein in the gastrocnemius ( a ) and soleus ( b ) of WT and bGH mice. Phosphorylation <t>of</t> <t>4E-BP1</t> and 4E-BP1 total protein in the gastrocnemius ( c ) and soleus ( d ) of WT and bGH mice. p70 S6 kinase phosphorylation and p70 S6 kinase total protein in the gastrocnemius ( e ) and soleus ( f ) of WT and bGH mice. Values in A.U. were normalized to a control sample run on each blot and then presented relative to GAPDH. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT control mice
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    1) Product Images from "Mice overexpressing growth hormone exhibit increased skeletal muscle myostatin and MuRF1 with attenuation of muscle mass"

    Article Title: Mice overexpressing growth hormone exhibit increased skeletal muscle myostatin and MuRF1 with attenuation of muscle mass

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-017-0133-y

    Phosphorylation and total protein involved in protein synthesis in the gastrocnemius and soleus of bGH ( n = 9) and WT ( n = 9) mice. mTOR phosphorylation and mTOR total protein in the gastrocnemius ( a ) and soleus ( b ) of WT and bGH mice. Phosphorylation of 4E-BP1 and 4E-BP1 total protein in the gastrocnemius ( c ) and soleus ( d ) of WT and bGH mice. p70 S6 kinase phosphorylation and p70 S6 kinase total protein in the gastrocnemius ( e ) and soleus ( f ) of WT and bGH mice. Values in A.U. were normalized to a control sample run on each blot and then presented relative to GAPDH. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT control mice
    Figure Legend Snippet: Phosphorylation and total protein involved in protein synthesis in the gastrocnemius and soleus of bGH ( n = 9) and WT ( n = 9) mice. mTOR phosphorylation and mTOR total protein in the gastrocnemius ( a ) and soleus ( b ) of WT and bGH mice. Phosphorylation of 4E-BP1 and 4E-BP1 total protein in the gastrocnemius ( c ) and soleus ( d ) of WT and bGH mice. p70 S6 kinase phosphorylation and p70 S6 kinase total protein in the gastrocnemius ( e ) and soleus ( f ) of WT and bGH mice. Values in A.U. were normalized to a control sample run on each blot and then presented relative to GAPDH. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT control mice

    Techniques Used:

    total 4e bp1  (Santa Cruz Biotechnology)

     
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    anti total 4e bp1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology anti total 4e bp1
    A. VRB disrupts eIF4F complex formation and <t>enhances</t> <t>eIF4E/4E-BP1</t> interactions. U2OS cells without (−) or with VRB (150 μM), SA (100 μM), DOX (150 μM) or hydrogen peroxide (H 2 O 2 , 1mM) treatment (1hour) were lysed and subjected to m 7 GTP-sepaharose pull down to isolate cap-bound complexes. Both the input (input) and precipitated (m7GTP) fractions were subjected to western blotting and probed against eIF4G, eIF4E and non-phosphorylated 4E-BP1 (non-ph-4EBP1). B. The same as in 5A, except that U2OS cells were treated with indicated VAs and levels of non-phosphorylated 4E-BP1 (non-ph-4EBP1) were compared to control treatment (−). Levels of eIF4G and eIF4E were served as controls. C. VRB treatment does not affect ph-RPS6 status. Whole cell lysates from VRB-, SA-, DMSO-treated and untreated (−) U2OS cells were subjected to western blotting using ph-eIF2α-, ph-RPS6 and non-ph-4E-BP1-specific antibodies. RACK1 was used as loading control. D. VRB-induced SG formation in MEFs carrying non-phosphorylatable variant of RPS6 (ph-RPS6 −/− ). WT and ph-RPS6 −/− MEFs were subjected to no stress (−), 100 μM SA and 150 μM VRB and stained with G3BP1 and Hoechst (nuclei). Size bar represents 10 μm. E. Depletion of 4E-BP1 inhibits VRB-induced SG formation. WT (no siRNA), control siRNA- (ctrl), 4E-BP1- and 4E-BP1/PERK-treated U2OS cells subjected to 100 μM SA and 150 μM VRB, stained with G3BP1 and % of SG-positive cells was determined. Standard statistics was applied (unpaired Student's t -test (p-values are shown, N = 3)).
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    1) Product Images from "Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation"

    Article Title: Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8728

    A. VRB disrupts eIF4F complex formation and enhances eIF4E/4E-BP1 interactions. U2OS cells without (−) or with VRB (150 μM), SA (100 μM), DOX (150 μM) or hydrogen peroxide (H 2 O 2 , 1mM) treatment (1hour) were lysed and subjected to m 7 GTP-sepaharose pull down to isolate cap-bound complexes. Both the input (input) and precipitated (m7GTP) fractions were subjected to western blotting and probed against eIF4G, eIF4E and non-phosphorylated 4E-BP1 (non-ph-4EBP1). B. The same as in 5A, except that U2OS cells were treated with indicated VAs and levels of non-phosphorylated 4E-BP1 (non-ph-4EBP1) were compared to control treatment (−). Levels of eIF4G and eIF4E were served as controls. C. VRB treatment does not affect ph-RPS6 status. Whole cell lysates from VRB-, SA-, DMSO-treated and untreated (−) U2OS cells were subjected to western blotting using ph-eIF2α-, ph-RPS6 and non-ph-4E-BP1-specific antibodies. RACK1 was used as loading control. D. VRB-induced SG formation in MEFs carrying non-phosphorylatable variant of RPS6 (ph-RPS6 −/− ). WT and ph-RPS6 −/− MEFs were subjected to no stress (−), 100 μM SA and 150 μM VRB and stained with G3BP1 and Hoechst (nuclei). Size bar represents 10 μm. E. Depletion of 4E-BP1 inhibits VRB-induced SG formation. WT (no siRNA), control siRNA- (ctrl), 4E-BP1- and 4E-BP1/PERK-treated U2OS cells subjected to 100 μM SA and 150 μM VRB, stained with G3BP1 and % of SG-positive cells was determined. Standard statistics was applied (unpaired Student's t -test (p-values are shown, N = 3)).
    Figure Legend Snippet: A. VRB disrupts eIF4F complex formation and enhances eIF4E/4E-BP1 interactions. U2OS cells without (−) or with VRB (150 μM), SA (100 μM), DOX (150 μM) or hydrogen peroxide (H 2 O 2 , 1mM) treatment (1hour) were lysed and subjected to m 7 GTP-sepaharose pull down to isolate cap-bound complexes. Both the input (input) and precipitated (m7GTP) fractions were subjected to western blotting and probed against eIF4G, eIF4E and non-phosphorylated 4E-BP1 (non-ph-4EBP1). B. The same as in 5A, except that U2OS cells were treated with indicated VAs and levels of non-phosphorylated 4E-BP1 (non-ph-4EBP1) were compared to control treatment (−). Levels of eIF4G and eIF4E were served as controls. C. VRB treatment does not affect ph-RPS6 status. Whole cell lysates from VRB-, SA-, DMSO-treated and untreated (−) U2OS cells were subjected to western blotting using ph-eIF2α-, ph-RPS6 and non-ph-4E-BP1-specific antibodies. RACK1 was used as loading control. D. VRB-induced SG formation in MEFs carrying non-phosphorylatable variant of RPS6 (ph-RPS6 −/− ). WT and ph-RPS6 −/− MEFs were subjected to no stress (−), 100 μM SA and 150 μM VRB and stained with G3BP1 and Hoechst (nuclei). Size bar represents 10 μm. E. Depletion of 4E-BP1 inhibits VRB-induced SG formation. WT (no siRNA), control siRNA- (ctrl), 4E-BP1- and 4E-BP1/PERK-treated U2OS cells subjected to 100 μM SA and 150 μM VRB, stained with G3BP1 and % of SG-positive cells was determined. Standard statistics was applied (unpaired Student's t -test (p-values are shown, N = 3)).

    Techniques Used: Western Blot, Variant Assay, Staining

    A. Viability of control siRNA-, PERK- and PERK/4E-BP1-depleted cells in the presence of various VRB concentrations (24 hours). Viability was determined by measuring the degree of cell death. The half maximal inhibitory concentrations (IC 50 ) of VRB are shown. B. U2OS cells depleted of PERK or 4E-BP1 were subjected to 60, 70, 80μM of VRB for 1 hour. Control siRNA-treated cells (ctrl) were used as control. Whole cell lysates from VRB and untreated (−) U2OS cells (ctrl, PERK and 4E-BP1) were subjected to Western Blotting using Caspase 3- (inactive) and cleaved form of Caspase 3 (active)-specific antibodies. RACK1 was used as loading control. C. Percentage of cells undergoing apoptosis was determined by immunofluorescence using cleaved Caspase 3-specific antibodies (red) on populations of control siRNA-treated, PERK- and 4E-BP1-depleted cells subjected to 40 or 80 μM VRB. Representative IF images are shown. Standard statistics were applied (unpaired Student's t -test (p-values are shown, N = 3)). D. VRB-induced SG formation in SG-competent (U2OS, ΔΔG3BP1/2+G3BP1, ΔΔG3BP1/2+ S149A) and SG-incompetent (ΔΔG3BP1/2, ΔΔG3BP1/2+S149E) cells. Representative images with no treatment (−), 50 μM and 150 μM VRB are shown. eIF4G was used as a SG marker. E. Viability of SG-competent and incompetent U2OS cells (Figure ) treated with indicated VRB concentrations (0-100 μM, 24h). F. The half maximal inhibitory concentrations (IC50) of VRB and viability of ΔΔG3BP1/2 cells reconstituted with SG-competent S149A and SG-incompetent S149E mutants (24 h). Standard statistics was applied (unpaired Student's t -test (p-values are shown, N = 3)).
    Figure Legend Snippet: A. Viability of control siRNA-, PERK- and PERK/4E-BP1-depleted cells in the presence of various VRB concentrations (24 hours). Viability was determined by measuring the degree of cell death. The half maximal inhibitory concentrations (IC 50 ) of VRB are shown. B. U2OS cells depleted of PERK or 4E-BP1 were subjected to 60, 70, 80μM of VRB for 1 hour. Control siRNA-treated cells (ctrl) were used as control. Whole cell lysates from VRB and untreated (−) U2OS cells (ctrl, PERK and 4E-BP1) were subjected to Western Blotting using Caspase 3- (inactive) and cleaved form of Caspase 3 (active)-specific antibodies. RACK1 was used as loading control. C. Percentage of cells undergoing apoptosis was determined by immunofluorescence using cleaved Caspase 3-specific antibodies (red) on populations of control siRNA-treated, PERK- and 4E-BP1-depleted cells subjected to 40 or 80 μM VRB. Representative IF images are shown. Standard statistics were applied (unpaired Student's t -test (p-values are shown, N = 3)). D. VRB-induced SG formation in SG-competent (U2OS, ΔΔG3BP1/2+G3BP1, ΔΔG3BP1/2+ S149A) and SG-incompetent (ΔΔG3BP1/2, ΔΔG3BP1/2+S149E) cells. Representative images with no treatment (−), 50 μM and 150 μM VRB are shown. eIF4G was used as a SG marker. E. Viability of SG-competent and incompetent U2OS cells (Figure ) treated with indicated VRB concentrations (0-100 μM, 24h). F. The half maximal inhibitory concentrations (IC50) of VRB and viability of ΔΔG3BP1/2 cells reconstituted with SG-competent S149A and SG-incompetent S149E mutants (24 h). Standard statistics was applied (unpaired Student's t -test (p-values are shown, N = 3)).

    Techniques Used: Western Blot, Immunofluorescence, Marker

    total 4e bp1  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology anti total 4e bp1
    EDL muscles were held at optimal length ( Lo ) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, <t>and</t> <t>4E-BP1.</t> The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.
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    antibodies against total 4e bp1  (Santa Cruz Biotechnology)
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    (A) Wild-type and DKO MEFs were pre-incubated in complete medium in the presence of additional NaCl at the concentrations indicated. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and total protein synthesis was then measured by the incorporation of [ 35 S]methionine (2 µCi/ml) for 1 h as described in Experimental. The data are the means of triplicate determinations and are expressed as counts per min incorporated per µg of protein ± S.E.M. Significances of differences between incubations ± Ku-0063794 were determined by unpaired t tests: * = p<0.005; N.S. = not significant. (B) Extracts from <t>4E-BP</t> wild-type and DKO cells were analysed for expression <t>of</t> <t>4E-BP1</t> by immunoblotting. Blots for GAPDH are also shown as loading controls. (C) Summary of % inhibition of protein synthesis by Ku-0063794 in 4E-BP wild-type (wt) and DKO cells as a function of the additional NaCl concentration.
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    total 4e bp1  (Santa Cruz Biotechnology)
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    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total <t>p70S6K,</t> <t>p-4E-BP1</t> (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.
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    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total <t>p70S6K,</t> <t>p-4E-BP1</t> (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.
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    4e bp1 total  (Santa Cruz Biotechnology)
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    Phosphorylation and total protein involved in protein synthesis in the gastrocnemius and soleus of bGH ( n = 9) and WT ( n = 9) mice. mTOR phosphorylation and mTOR total protein in the gastrocnemius ( a ) and soleus ( b ) of WT and bGH mice. Phosphorylation <t>of</t> <t>4E-BP1</t> and 4E-BP1 total protein in the gastrocnemius ( c ) and soleus ( d ) of WT and bGH mice. p70 S6 kinase phosphorylation and p70 S6 kinase total protein in the gastrocnemius ( e ) and soleus ( f ) of WT and bGH mice. Values in A.U. were normalized to a control sample run on each blot and then presented relative to GAPDH. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT control mice
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    EDL muscles were held at optimal length ( Lo ) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Journal: PLoS ONE

    Article Title: Mechanical Stimulation Induces mTOR Signaling via an ERK-Independent Mechanism: Implications for a Direct Activation of mTOR by Phosphatidic Acid

    doi: 10.1371/journal.pone.0047258

    Figure Lengend Snippet: EDL muscles were held at optimal length ( Lo ) in an ex-vivo organ culture system and pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min. The muscles were then subjected to 15 or 90 min of intermittent 15% stretch or held static at Lo as a control condition. Muscles were collected at the end of the 15 or 90 min interval and subjected to western blot analysis for phosphorylated (P) and total ERK, p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the time-matched vehicle control samples (U0126 –, Stretch –). All values are presented as the mean (n = 3−6 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Article Snippet: Rabbit anti-phospho-p70 s6k (Thr389) and anti-total 4E-BP1 (R-113, used only for ) were purchased from Santa Cruz Biotechnologies (Santa Cruz,CA, USA).

    Techniques: Ex Vivo, Organ Culture, Incubation, Western Blot

    ( A ) EDL muscles from wild type mice, and ( B ) transgenic mice with muscle specific expression of a rapamycin-resistant mutant of mTOR (RR-mTOR), were held at Lo and pre-incubated with 150 nM rapamycin (RAP +) or the vehicle (RAP –, DMSO) for 30 min. The muscles were then subjected to 90 min of stretch or control conditions followed by western blot analysis for phosphorylated (P) and total p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the genotype-matched vehicle control samples (RAP–, Stretch–). Note: total 4E-BP1 gels were also run under conditions that minimize the gel mobility shift (short-run). All values are presented as the mean (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Journal: PLoS ONE

    Article Title: Mechanical Stimulation Induces mTOR Signaling via an ERK-Independent Mechanism: Implications for a Direct Activation of mTOR by Phosphatidic Acid

    doi: 10.1371/journal.pone.0047258

    Figure Lengend Snippet: ( A ) EDL muscles from wild type mice, and ( B ) transgenic mice with muscle specific expression of a rapamycin-resistant mutant of mTOR (RR-mTOR), were held at Lo and pre-incubated with 150 nM rapamycin (RAP +) or the vehicle (RAP –, DMSO) for 30 min. The muscles were then subjected to 90 min of stretch or control conditions followed by western blot analysis for phosphorylated (P) and total p70, S6, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the genotype-matched vehicle control samples (RAP–, Stretch–). Note: total 4E-BP1 gels were also run under conditions that minimize the gel mobility shift (short-run). All values are presented as the mean (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Article Snippet: Rabbit anti-phospho-p70 s6k (Thr389) and anti-total 4E-BP1 (R-113, used only for ) were purchased from Santa Cruz Biotechnologies (Santa Cruz,CA, USA).

    Techniques: Transgenic Assay, Expressing, Mutagenesis, Incubation, Western Blot, Mobility Shift

    ( A ) EDL muscles were held at Lo and pre-labeled with [ 3 H]-myristic acid for 2 h. The muscles were then subjected to 15 or 90 min of stretch or control conditions. Muscles were collected at the end of the 15 or 90 min interval, and the amount of 3 H-labeled PA ( 3 H PA) was measured and then expressed as a percentage of time-matched control values. The values are presented as the mean + SEM (n = 4−7 per group). • Significantly different from the time-matched control group. ( B ) C2C12 myoblasts were serum-starved overnight and then pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min, followed by 20 min stimulation with 30 µM C8 PA, 300 µM Egg PA or the vehicle (Control, PBS). The samples were then subjected to western blot analysis for phosphorylated (P) and total ERK, p70, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the vehicle control samples (U0126 –, PBS). All values are presented as the mean and were obtained from three independent experiments (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Journal: PLoS ONE

    Article Title: Mechanical Stimulation Induces mTOR Signaling via an ERK-Independent Mechanism: Implications for a Direct Activation of mTOR by Phosphatidic Acid

    doi: 10.1371/journal.pone.0047258

    Figure Lengend Snippet: ( A ) EDL muscles were held at Lo and pre-labeled with [ 3 H]-myristic acid for 2 h. The muscles were then subjected to 15 or 90 min of stretch or control conditions. Muscles were collected at the end of the 15 or 90 min interval, and the amount of 3 H-labeled PA ( 3 H PA) was measured and then expressed as a percentage of time-matched control values. The values are presented as the mean + SEM (n = 4−7 per group). • Significantly different from the time-matched control group. ( B ) C2C12 myoblasts were serum-starved overnight and then pre-incubated with 50 µM U0126 (U0126+) or the vehicle (U0126 –, DMSO) for 30 min, followed by 20 min stimulation with 30 µM C8 PA, 300 µM Egg PA or the vehicle (Control, PBS). The samples were then subjected to western blot analysis for phosphorylated (P) and total ERK, p70, and 4E-BP1. The total amount, and phospho:total ratios, of each protein were measured and then expressed relative to the values obtained in the vehicle control samples (U0126 –, PBS). All values are presented as the mean and were obtained from three independent experiments (n = 3−5 per group). *Significantly different from the drug-matched control group, †Significantly different from the stimulation-matched vehicle group, P≤0.05.

    Article Snippet: Rabbit anti-phospho-p70 s6k (Thr389) and anti-total 4E-BP1 (R-113, used only for ) were purchased from Santa Cruz Biotechnologies (Santa Cruz,CA, USA).

    Techniques: Labeling, Incubation, Western Blot

    (A) Wild-type and DKO MEFs were pre-incubated in complete medium in the presence of additional NaCl at the concentrations indicated. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and total protein synthesis was then measured by the incorporation of [ 35 S]methionine (2 µCi/ml) for 1 h as described in Experimental. The data are the means of triplicate determinations and are expressed as counts per min incorporated per µg of protein ± S.E.M. Significances of differences between incubations ± Ku-0063794 were determined by unpaired t tests: * = p<0.005; N.S. = not significant. (B) Extracts from 4E-BP wild-type and DKO cells were analysed for expression of 4E-BP1 by immunoblotting. Blots for GAPDH are also shown as loading controls. (C) Summary of % inhibition of protein synthesis by Ku-0063794 in 4E-BP wild-type (wt) and DKO cells as a function of the additional NaCl concentration.

    Journal: PLoS ONE

    Article Title: Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

    doi: 10.1371/journal.pone.0071138

    Figure Lengend Snippet: (A) Wild-type and DKO MEFs were pre-incubated in complete medium in the presence of additional NaCl at the concentrations indicated. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and total protein synthesis was then measured by the incorporation of [ 35 S]methionine (2 µCi/ml) for 1 h as described in Experimental. The data are the means of triplicate determinations and are expressed as counts per min incorporated per µg of protein ± S.E.M. Significances of differences between incubations ± Ku-0063794 were determined by unpaired t tests: * = p<0.005; N.S. = not significant. (B) Extracts from 4E-BP wild-type and DKO cells were analysed for expression of 4E-BP1 by immunoblotting. Blots for GAPDH are also shown as loading controls. (C) Summary of % inhibition of protein synthesis by Ku-0063794 in 4E-BP wild-type (wt) and DKO cells as a function of the additional NaCl concentration.

    Article Snippet: Antibodies against total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ) were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany) and Cell Signalling Technology (Hitchin, Herts, UK) respectively.

    Techniques: Incubation, Expressing, Western Blot, Inhibition, Concentration Assay

    4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. The extracts were analysed for total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ), total and phosphorylated Akt (Ser 473 ), and total and phosphorylated p70S6kinase (Thr 421 /Ser 424 and Thr 389 ) by SDS gel electrophoresis and immunoblotting. The positions of the differentially phosphorylated α, β and γ forms and of a cleavage product of 4E-BP1 are indicated. Blots for GAPDH are also shown as loading controls.

    Journal: PLoS ONE

    Article Title: Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

    doi: 10.1371/journal.pone.0071138

    Figure Lengend Snippet: 4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. The extracts were analysed for total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ), total and phosphorylated Akt (Ser 473 ), and total and phosphorylated p70S6kinase (Thr 421 /Ser 424 and Thr 389 ) by SDS gel electrophoresis and immunoblotting. The positions of the differentially phosphorylated α, β and γ forms and of a cleavage product of 4E-BP1 are indicated. Blots for GAPDH are also shown as loading controls.

    Article Snippet: Antibodies against total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ) were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany) and Cell Signalling Technology (Hitchin, Herts, UK) respectively.

    Techniques: Incubation, SDS-Gel, Electrophoresis, Western Blot

    4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. Using equal quantities of total protein, the extracts were then subjected to affinity chromatography on m 7 GTP-Sepharose to isolate eIF4E and its associated proteins, as described in Experimental. The bound proteins were analysed for eIF4E, 4E-BP1 and eIF4GI by SDS gel electrophoresis and immunoblotting.

    Journal: PLoS ONE

    Article Title: Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

    doi: 10.1371/journal.pone.0071138

    Figure Lengend Snippet: 4E-BP wild-type and DKO MEFs were pre-incubated in complete medium in the absence or presence of additional 0.1 M NaCl. After 1 h the cells were further incubated with or without Ku-0063794 (1 µM) for 1 h and extracts were prepared. Using equal quantities of total protein, the extracts were then subjected to affinity chromatography on m 7 GTP-Sepharose to isolate eIF4E and its associated proteins, as described in Experimental. The bound proteins were analysed for eIF4E, 4E-BP1 and eIF4GI by SDS gel electrophoresis and immunoblotting.

    Article Snippet: Antibodies against total 4E-BP1 and phosphorylated 4E-BP1 (Ser 64 ) were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany) and Cell Signalling Technology (Hitchin, Herts, UK) respectively.

    Techniques: Incubation, Affinity Chromatography, SDS-Gel, Electrophoresis, Western Blot

    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.

    Journal: PLoS ONE

    Article Title: Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

    doi: 10.1371/journal.pone.0103071

    Figure Lengend Snippet: HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. * P <0.05 versus the control, # P <0.01 versus the control.

    Article Snippet: Primary antibodies against the following proteins were used: CD36, total p70S6K, p-p70S6K (Thr 421/Ser 424), total 4E-BP1, p-4E-BP1 (Ser 65/Thr 70), total eIF4E, p-eIF4E (Ser 209) and β-actin (Santa Cruz, Dallas, TX, USA); total mTOR (Millipore, Temecula, CA, USA); and p-mTOR (phospho S2448) (Abcam, Cambridge, UK).

    Techniques: Incubation, Injection, Western Blot

    HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (A) and livers (B). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. A polysomal analysis was performed to detect CD36 translational efficiency in the cells (C) and livers (D). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at P <0.05, and # denotes a significant difference at P <0.01.

    Journal: PLoS ONE

    Article Title: Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

    doi: 10.1371/journal.pone.0103071

    Figure Lengend Snippet: HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (A) and livers (B). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. A polysomal analysis was performed to detect CD36 translational efficiency in the cells (C) and livers (D). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at P <0.05, and # denotes a significant difference at P <0.01.

    Article Snippet: Primary antibodies against the following proteins were used: CD36, total p70S6K, p-p70S6K (Thr 421/Ser 424), total 4E-BP1, p-4E-BP1 (Ser 65/Thr 70), total eIF4E, p-eIF4E (Ser 209) and β-actin (Santa Cruz, Dallas, TX, USA); total mTOR (Millipore, Temecula, CA, USA); and p-mTOR (phospho S2448) (Abcam, Cambridge, UK).

    Techniques: Incubation, Injection, Western Blot

    Phosphorylation and total protein involved in protein synthesis in the gastrocnemius and soleus of bGH ( n = 9) and WT ( n = 9) mice. mTOR phosphorylation and mTOR total protein in the gastrocnemius ( a ) and soleus ( b ) of WT and bGH mice. Phosphorylation of 4E-BP1 and 4E-BP1 total protein in the gastrocnemius ( c ) and soleus ( d ) of WT and bGH mice. p70 S6 kinase phosphorylation and p70 S6 kinase total protein in the gastrocnemius ( e ) and soleus ( f ) of WT and bGH mice. Values in A.U. were normalized to a control sample run on each blot and then presented relative to GAPDH. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT control mice

    Journal: Skeletal Muscle

    Article Title: Mice overexpressing growth hormone exhibit increased skeletal muscle myostatin and MuRF1 with attenuation of muscle mass

    doi: 10.1186/s13395-017-0133-y

    Figure Lengend Snippet: Phosphorylation and total protein involved in protein synthesis in the gastrocnemius and soleus of bGH ( n = 9) and WT ( n = 9) mice. mTOR phosphorylation and mTOR total protein in the gastrocnemius ( a ) and soleus ( b ) of WT and bGH mice. Phosphorylation of 4E-BP1 and 4E-BP1 total protein in the gastrocnemius ( c ) and soleus ( d ) of WT and bGH mice. p70 S6 kinase phosphorylation and p70 S6 kinase total protein in the gastrocnemius ( e ) and soleus ( f ) of WT and bGH mice. Values in A.U. were normalized to a control sample run on each blot and then presented relative to GAPDH. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT control mice

    Article Snippet: Muscle lysate (20–30 μg cellular protein) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA) and probed overnight with Cell Signaling (Beverly, MA) antibodies for JAK2 (Tyr1007/1008), JAK2 total, STAT5 (Tyr694), STAT5 total, Akt1 (Ser473), Akt2 (Ser474), Akt1 total, Akt2 total, AS160 (Ser588), AS160 (Ser666), AS160 total, TBC1D1 (Thr590), TBC1D1 (Ser700), TBC1D1 total, insulin receptor β, IRS1 (Ser307), mTOR (Ser2448), mTOR total, p70 S6 kinase (Thr389), p70 S6 kinase total, 4E-BP1 (Thr37/46), 4E-BP1 total, p38 (Thr180/Tyr182), p38 total, p44/42 (Thr202/Tyr204), p44/42 total, JNK (Thr183/Tyr185), JNK total, GSK-3β (Ser9), GSK3α/β total, Fox01 (Thr24)/Fox03a (Thr32), FOXO1 total, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Santa Cruz Biotechnology (Santa Cruz, CA) antibodies for IRS1 total, myostatin, and muscle RING factor 1 (MuRF1), Developmental Studies Hybridoma Bank (University of Iowa) antibodies for myosin heavy chain (MHC) I and MHC IIb, as well as, an Abcam (Cambridge, MA) antibody for Activin Receptor Type IIB.

    Techniques: