Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hyperphosphorylation of ribosomal protein S6 predicts unfavorable clinical survival in non-small cell lung cancer

doi: 10.1186/s13046-015-0239-1

Figure Lengend Snippet: The upstream regulation signaling pathway of rpS6. a Separate overexpression of Akt1, Akt2 and Akt3 in HBE cells showed that only Akt2 (oe-Akt2) led to marked hyperphosphorylation of mTOR, p70S6K and rpS6 (left; All P < 0.05); while Akt1 or Akt3 alteration resulted in no detectable change of these proteins (left; All P > 0.05). In H1650 and SK-MES-1 cell lines, only the specific silence of Akt2 (sh-Akt2) caused significant loss of p-mTOR, p-p70S6K and p-rpS6 (middle and right; All P < 0.05), and no notable alteration with the Akt1 and Akt3 knockdown were found (sh-Akt1, sh-Akt3; middle and right; All P < 0.05). b H1650 and SK-MES-1 were treated with Akti-2 (Akt inhibitor XII) for 72 h in concentration of 0.8, 3.2 and 12.8 μM. Western blot assays revealed the strongest effect of 12.8 μM and the accompanied dephosphorylation of mTOR, p70S6K and rpS6 (All P < 0.05). oe-Akt1: overexpression of Akt1; oe-NCa1: negative control for oe-Akt1; oe-Akt2: overexpression of oe-Akt2; oe-NCa2: negative control for oe-Akt2; oe-Akt3: overexpression of oe-Akt3; oe-NCa3: negative control for oe-Akt3. sh-Akt1: knockdown for Akt1; sh-NCa1: negative control for sh-Akt1; sh-Akt2: knockdown for Akt2; sh-NCa2: negative control for sh-Akt2; sh-Akt3: knockdown for Akt3; sh-NCa3: negative control for sh-Akt3

Article Snippet: Specific protein antibodies included anti-total rpS6 (CST, #2217), anti-p-rpS6 (Ser235/236, CST, #4858), anti-p21 Cip1 (CST, #2947), anti-p27 Kip1 (CST, #3688), anti-CDK2 (CST, #2546), anti-CDK4 (CST, #12790), anti-Cyclin E (SAB, #29030), anti-cyclin D1 (CST, #2978), anti-p-Rb (Ser780, CST, #3590), anti-Paxillin (CST, #12065), anti-p-Paxillin (Tyr118; CST, #2541), anti-vimentin (CST, #5741), anti-N-cadherin (CST, #4061), anti-E-cadherin (CST, #3195), anti-MMP-9 (CST, #13667), anti-MMP-2 (CST, # 5741), anti-t-Akt1 (CST, #2967), anti-p-Akt1 (Ser473, CST, # 9018), anti-t-Akt2 (CST, #2964), anti-p-Akt2 (Ser474, CST, #8599), anti-t-Akt3 (Biorbyt, #orb6787), anti-p-Akt3 (Ser472, Biorbyt, #orb6790), anti-t-mTOR (CST, #2983), anti-p-mTOR (Ser2448, CST, #5536), anti-t-p70S6K (SAB, #21276) and anti-p-p70S6K (Ser424, SAB, #21276) and anti-β-actin (CST, #4970) as an internal control.

Techniques: Over Expression, Concentration Assay, Western Blot, De-Phosphorylation Assay, Negative Control

Journal: Cells

Article Title: Central Suppression of the GH/IGF Axis and Abrogation of Exercise-Related mTORC1/2 Activation in the Muscle of Phenotype-Selected Male Marathon Mice (DUhTP)

doi: 10.3390/cells10123418

Figure Lengend Snippet: Effects of phenotype selection and endurance exercise on protein expression, phosphorylation, and specific activation of ( a ) PTEN and ( b ) p85S6K (left panel) and p70S6K (right panel) by Western immunoblot analysis in Musculus rectus femoris . The protein abundances in trained and untrained DUhTP (light/dark blue) and DUC mice (gray/black) were calculated relative to DUC sed (100%). Results are shown as box plots. Statistical analysis was performed using one-way ANOVA. Significant differences are marked with: * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviation: sed = sedentary.

Article Snippet: Antibodies detecting total S6K (CST: #2708S) or phosphorylated S6K (CST: #9234S) were used at a dilution of 1:1000 in 3% bovine serum albumin (BSA).

Techniques: Selection, Expressing, Phospho-proteomics, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Amelioration of Behavioral Abnormalities in BH 4 -deficient Mice by Dietary Supplementation of Tyrosine

doi: 10.1371/journal.pone.0060803

Figure Lengend Snippet: (A) Immunoblotting was performed on the cerebral homogenates separated from brains of 35-d-old Spr +/+ or Spr −/− mice. Each group of experimental mice was fed a normal diet (ND) or treated with the dietary tyrosine, or L-DOPA therapy for 10 days. Phosphorylation of S6 and S6K in the brain homogenates was examined to evaluate mTORC1 activity. The representative Western blot demonstrating mTORC1 recovery in Spr −/− mice by dietary tyrosine supplementation is shown. (B) The ratios of band intensities of pS6K/S6K and pS6/S6 were quantified by using image J software. Values are means ± SD (n ≥4 for each group of experimental mice). * P<0.05, ** P<0.01.

Article Snippet: Cerebral or midbrain proteins obtained from 35-d-old experimental mice were separated by 10% SDS-PAGE, blotted onto a nitrocellulose membrane, blocked with 5% BSA, incubated with anti-phospho S6K antibody (Thr389, #9234, 1∶500, Cell Signaling Technology), anti-S6K antibody (SC-8414, 1∶1,000, Santa Cruz Biotechnology), anti-phospho S6 antibody (Ser235/236, #2211, 1∶1,000, Cell Signaling Technology), anti-S6 antibody (#2317, 1∶500, Cell Signaling Technology), anti-TH antibody (1∶3,000, Millipore) or anti-actin antibody (SC1616, 1∶1,000, Santa Cruz Biotechnology), followed by an additional incubation with horseradish peroxide-conjugated anti-rabbit-IgG or anti-mouse-IgG antibodies (Jackson Laboratory).

Techniques: Western Blot, Phospho-proteomics, Activity Assay, Software

Journal: PLoS Biology

Article Title: Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling

doi: 10.1371/journal.pbio.3001432

Figure Lengend Snippet: Absorbance profile at 254 nm (A 254 ) and western blot analysis of fractionated cytoplasmic extracts from hippocampal tissue incubated in the absence or presence of MgCl 2 or RNase or EDTA. Monosome (80S), 60S ribosome, and polysome fractions are as indicated. Western blots performed from tri-chloroacetic acid–precipitated fractions to determine the distribution of the translation regulators eIF4E and p70 S6K; α7 subunit of the 20S core of the proteasome, Rpt1, Rpt3, Rpt6 of the 19S cap, and miRISC proteins MOV10 and Trim32 in the presence or absence of MgCl 2 or RNase or EDTA. (A) A 254 profile in the presence of MgCl 2 . (B) Western blots of the fractions obtained in (A). (C) A 254 profile obtained in the presence of RNase. (D) Western blots of the fractions obtained in (C). (E) A 254 profile in the presence of EDTA. (F) Western blots of the fractions obtained in (E). Rpt3 blots with different exposures are distinguished by a vertical black line to denote they represent separate panels within the figure. Two blots with different exposures are shown in the main figure and raw data to visualize the specific band of Rpt3. (G) A 254 profile of fractionated cytoplasmic extract used for determining activity of proteasomes. (H) Quantitation of catalytic activity of proteasomes present in alternate fractions from two polysome preparations. See also . The data underlying this figure are available at https://figshare.com/articles/dataset/Homeostatic_scaling_is_driven_by_a_translation-dependent_degradation_axis_that_recruits_miRISC_remodeling/16768816 . miRISC, miRNA-induced silencing complex; p70 S6K, p70 S6 kinase.

Article Snippet: Equal volumes of lysates were resolved on 8% to 10% SDS-PAGE, transferred onto nitrocellulose membrane, blocked with 5% BSA, and probed with antibodies against MOV10, Trim32, Ago, Dicer, Arg3.1 (CST, 1:250), p70 S6K, and phospho-p70 S6K.

Techniques: Western Blot, Incubation, Activity Assay, Quantitation Assay

Journal: PLoS Biology

Article Title: Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling

doi: 10.1371/journal.pbio.3001432

Figure Lengend Snippet: (A) Proteasome-associated protein complex was immunoprecipitated from hippocampal lysate using antibody against Rpt6 or mouse IgG. Western blot of purified protein complex performed using antibodies against Rpt6, eEF2, p70 S6K, and phospho-p70 S6K. (B) RiboTag mouse when crossed with CamKIIa promoter-driven Cre recombinase mouse results in the deletion of wild-type Rpl22 ribosomal protein and replacement of HA-tagged Rpl22 in forebrain excitatory neurons. (C) A 254 profile showing indicated fractions of monosome and polysome. (D) Polysome fractions from (C) showing enrichment of HA-Rpl22 as detected by western blot using antibody against HA. (E) HA-tagged Rpl22 containing polyribosome was affinity-purified using antibody against HA. Western blot analysis of affinity-purified complex shows the presence of HA, Rpt6, Trim32, and MOV10. See also . (F) MOV10 immunoprecipitated from hippocampal lysates. Western blot analysis of MOV10-immunoprecipitated protein complex shows the coprecipitation of Trim32 with miRISC components MOV10 and Ago. (G) Detection of HspA2 and Rpt6 in HA affinity-purified protein complex from HA-Rpl22 expressing neurons by western blot using antibody against HspA2, Rpt6 and HA. (H, I) A 254 profile showing indicated fractions of monosome and polysome obtained from cytoplasmic lysates treated with (H) or without (I) RNase prior to density gradient fractionation. See also . ( J ) HA-tagged Rpl22 containing ribosomes affinity-purified from heavy fractions of sucrose gradient using antibody against HA. Western blot analysis of affinity-purified complex with antibodies against HA, p70 S6K, eIF4E, 20S Core subunits, Rpt6, and Rpt1. The data underlying this figure are available at https://figshare.com/articles/dataset/Homeostatic_scaling_is_driven_by_a_translation-dependent_degradation_axis_that_recruits_miRISC_remodeling/16768816 . Ago, Argonaute; HA, haemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitation; miRISC, miRNA-induced silencing complex; p70 S6K, p70 S6 kinase; WT, wild-type.

Article Snippet: Equal volumes of lysates were resolved on 8% to 10% SDS-PAGE, transferred onto nitrocellulose membrane, blocked with 5% BSA, and probed with antibodies against MOV10, Trim32, Ago, Dicer, Arg3.1 (CST, 1:250), p70 S6K, and phospho-p70 S6K.

Techniques: Immunoprecipitation, Western Blot, Purification, Affinity Purification, Expressing, Fractionation

Journal: PLoS Biology

Article Title: Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling

doi: 10.1371/journal.pbio.3001432

Figure Lengend Snippet: Cortical neurons were treated with bicuculline or vehicle for 24 hours and then subjected to polysome fractionation. Western blot analysis was performed from the tri-chloroacetic acid–precipitated fractions to determine the distribution of translation regulators, miRISC components, chaperone, and the proteasome subunits in the fractions. (A, B) A 254 profile of fractionated cytoplasmic extracts from cortical neurons treated with vehicle (A) or bicuculline (B). Monosome (80S) or polysome fractions as indicated. (C, D) Western blot analysis of fractions from vehicle (C) or bicuculline (D) treated neurons showing the distribution of translation regulators eIF4E, eEF2, Rp S6, ph-Rp S6, p70 S6K, ph-p70 S6K; chaperone protein HspA2; proteasome subunit of 20S core, and Rpt1, Rpt3, Rpt6 of 19S cap, miRISC proteins Ago, MOV10, and Trim32. Rpt1 blots with different exposures are distinguished by a vertical black line to denote that they represent separate panels within the figure. Two blots with different exposures are shown in the main figure and raw data to visualize the specific band of Rpt1. ( E ) Quantitation of polysome distribution of Trim32. ( F ) Quantitation of polysome distribution of MOV10. * p < 0.005, ** p < 0.002. n = 3, Unpaired t test with Welch’s correction. See also . The data underlying this figure are available at https://figshare.com/articles/dataset/Homeostatic_scaling_is_driven_by_a_translation-dependent_degradation_axis_that_recruits_miRISC_remodeling/16768816 . Ago, Argonaute; miRISC, miRNA-induced silencing complex; ph-Rp S6, phosphorylated ribosomal protein S6; ph-p70 S6K, phosphorylated p70 S6 kinase; p70 S6K, p70 S6 kinase; RNP, ribonucleoprotein; Rp S6, ribosomal protein S6.

Article Snippet: Equal volumes of lysates were resolved on 8% to 10% SDS-PAGE, transferred onto nitrocellulose membrane, blocked with 5% BSA, and probed with antibodies against MOV10, Trim32, Ago, Dicer, Arg3.1 (CST, 1:250), p70 S6K, and phospho-p70 S6K.

Techniques: Fractionation, Western Blot, Quantitation Assay

Journal: PLoS Biology

Article Title: Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling

doi: 10.1371/journal.pbio.3001432

Figure Lengend Snippet: (A) Western blot analysis from neurons treated with bicuculline, rapamycin, or both showing the phosphorylation and total expression of p70 S6K and 4E-BP2. (B) Quantitation of p70 S6K phosphorylation. (C) Quantitation of 4E-BP2 phosphorylation. n = 4. * p < 0.0001. ns, not significant. Data shown as mean ± SEM. One-way ANOVA and Fisher’s LSD. (D) Western blot analysis from neurons treated with bicuculline, p70 S6K inhibitor LY2584702 Tosylate, or both, showing the expression of MOV10 and Trim32. (E) Quantitation of Trim32 expression. (F) Quantitation of MOV10 expression. n = 5. * p < 0.003, ** p < 0.0002 (E) ** p < 0.0001, * p < 0.02 (F). ns, not significant. Data shown as mean ± SEM. One-way ANOVA and Fisher’s LSD. The data underlying this figure are available at https://figshare.com/articles/dataset/Homeostatic_scaling_is_driven_by_a_translation-dependent_degradation_axis_that_recruits_miRISC_remodeling/16768816 . mTORC1, mammalian Target Of Rapamycin Complex-1; ns, not significant; p70 S6K, p70 S6 kinase; 4E-BP2, 4E-binding protein 2.

Article Snippet: Equal volumes of lysates were resolved on 8% to 10% SDS-PAGE, transferred onto nitrocellulose membrane, blocked with 5% BSA, and probed with antibodies against MOV10, Trim32, Ago, Dicer, Arg3.1 (CST, 1:250), p70 S6K, and phospho-p70 S6K.

Techniques: Western Blot, Phospho-proteomics, Expressing, Quantitation Assay, Binding Assay

Journal: PLoS Biology

Article Title: Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling

doi: 10.1371/journal.pbio.3001432

Figure Lengend Snippet: Reagents and resources.

Article Snippet: Equal volumes of lysates were resolved on 8% to 10% SDS-PAGE, transferred onto nitrocellulose membrane, blocked with 5% BSA, and probed with antibodies against MOV10, Trim32, Ago, Dicer, Arg3.1 (CST, 1:250), p70 S6K, and phospho-p70 S6K.

Techniques: Purification, Control, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, Luciferase, Transgenic Assay, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: mTORC1 deregulation and increased invasiveness cohere with dispersed endolysosomes in high-grade bladder cancer

doi: 10.1101/2020.07.10.196931

Figure Lengend Snippet: A. Western Blot analysis of eIF4E Binding Protein (4EBP1) phosphorylation (P-4EBP1 Ser65) in MGHU3, RT112, KU19-19 and JMSU1 and quantification of relative phosphorylated P-4EBP1 to GAPDH and total 4EBP1 protein levels. Error bars show s.d. of three independent experiments. B. Western Blot analysis of p70-S6 Kinase 1 (S6K1) phosphorylation (P-p70-S6K Thr389) in MGHU3, RT112, KU19-19 and JMSU1 and quantification of relative phosphorylated P-4EBP1 to GAPDH and total S6K1 protein levels. Error bars show s.d. of three independent experiments. C. Representative images of MGHU3, RT112, KU19-19 and JMSU1 cells transfected with TFEB-GFP (green) for 48h and quantification of relative fluorescent intensity between nucleus (grey) and cytoplasm (black). Scale bars are 10 μm. Error bars show s.d. of three independent experiments. D. Representative images of endolysosomes visualized by immunofluorescence staining against the lysosomal-associated membrane protein 1 (LAMP-1, CD107a) and mTORC1 visualized by immunofluorescence staining against mTOR in MGHU3, RT112, KU19-19 and JMSU1. The zoom shows the merged image of both proteins. Scale bars are 10 μm.

Article Snippet: Concentration of the primary antibody used were as follows: Phospho P-70 (Thr389)-S6K (CST: 9205S, 1:1000 in 5% BSA in TBST), P-70 S6K (CST: 9202S, 1:1000 in 5% milk in TBST), Phospho(Ser65)-4EBP1 (CST: 9451, 1:1000 in 5% BSA in TBST), 4EBP1(CST: 9452, 1:1000 in 5% milk in TBST), GAPDH (Sigma: G9545, 1:10,000 in 5% milk in TBST).

Techniques: Western Blot, Binding Assay, Phospho-proteomics, Transfection, Immunofluorescence, Staining, Membrane

Journal: bioRxiv

Article Title: mTORC1 deregulation and increased invasiveness cohere with dispersed endolysosomes in high-grade bladder cancer

doi: 10.1101/2020.07.10.196931

Figure Lengend Snippet: A. Western Blot analysis of eIF4E Binding Protein (4EBP1) phosphorylation (P-4EBP1 Ser65) and p70-S6 Kinase 1 (S6K1) phosphorylation (P-p70-S6K Thr389) in MGHU3, RT112, KU19-19 and JMSU1 in control conditions (full media) and after treatment with Wortmannin at 1μM for 2 h. B. Western Blot analysis of 4EBP1 and S6K phosphorylation in MGHU3, RT112, KU19-19 and JMSU1 in control conditions (full media) and after treatment with Rapamycin at 20μM for 2 h. C. Western Blot analysis of 4EBP1 and S6K phosphorylation in MGHU3, RT112, KU19-19 and JMSU1 in control conditions (full media) and after treatment with Torin at 1μM for 2 h. D. Western Blot analysis of 4EBP1 and S6K phosphorylation in MGHU3, RT112, KU19-19 and JMSU1 in control conditions (full media) and after starvation in EBSS for 4h. E. Representative images of MGHU3, RT112, KU19-19 and JMSU1 cells transfected with TFEB-GFP (green) for 48h and treated with Rapamycin at 20μM for 2 h. Scale bars are 5 μm.

Article Snippet: Concentration of the primary antibody used were as follows: Phospho P-70 (Thr389)-S6K (CST: 9205S, 1:1000 in 5% BSA in TBST), P-70 S6K (CST: 9202S, 1:1000 in 5% milk in TBST), Phospho(Ser65)-4EBP1 (CST: 9451, 1:1000 in 5% BSA in TBST), 4EBP1(CST: 9452, 1:1000 in 5% milk in TBST), GAPDH (Sigma: G9545, 1:10,000 in 5% milk in TBST).

Techniques: Western Blot, Binding Assay, Phospho-proteomics, Control, Transfection