Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Adult-onset repression of S6K ubiquitously using actGS>S6K RNAi extended lifespan (n=200). b , Adult-onset repression of S6K in the fat body using Lsp2GS>S6K RNAi extended lifespan (n=180). c , Rapamycin extended lifespan of control flies, but not of flies with ubiquitous overexpression of constitutively active S6K ( daGS>S6K CA ). Ubiquitous overexpression of constitutively active S6K significantly attenuated the response to rapamycin treatment (rapamycin: p<0.0001, daGS>S6K CA induction: p<0.0001, interaction p=0.0236, n=200). d , Adult-onset S6K activation in the fat body ( Lsp2GS>S6K CA ) significantly attenuated rapamycin-related longevity (rapamycin: p<0.0001, Lsp2GS>S6K CA induction: p<0.0001, interaction p=0.0331, n=200). Log-rank test and Cox Proportional Hazards (CPH) test.

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Over Expression, Activation Assay

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Lysotracker staining of fat bodies from young (day 10) flies treated with rapamycin and overexpressing constitutively active S6K ( Lsp2GS>S6K CA ). Fat body-specific adult-onset overexpression of constitutively active S6K significantly increased acidic organelle size in response to rapamycin treatment (rapamycin: p<0.0001, Lsp2GS>S6K CA induction: p<0.0001, interaction p=0.0246, n=12). b , Lysotracker-positive enlarged acidic organelles (red) colocalized with the lysosomal marker Lamp1 ( Lsp2GS>S6K CA ; GFP-Lamp1 , green, upper panel) and partially colocalized with Rab7 ( Lsp2GS>S6K CA ; YFP-Rab7 , green, lower panel), a marker for late endosomes, in young flies treated with rapamycin and overexpressing S6K CA . c , Electron microscopy imaging of fat bodies of young flies treated with rapamycin and overexpressing S6K CA . Overexpression of S6K CA in the fat body attenuated the effect of rapamycin on multilamellar lysosomes (rapamycin: p=0.0395, Lsp2GS>S6K CA induction: p=0.2798, interaction p=0.0388, n=5). d , Lysotracker staining of young fat bodies overexpressing dominant negative Rab7 ( Lsp2GS>Rab7 DN ). Fat body-specific overexpression of Rab7 DN significantly increased acidic organelle size (p=0.0064, n=12). e , Fat body-specific overexpression of dominant negative Rab5 ( Lsp2GS>Rab5 DN ) also increased acidic organelle size (p=0.0798, n=12). Data are displayed as Tukey box plot (a, d-e) or mean□±□s.e.m. (c ). Each data point represents an average value per fat body. Scale bar, 10 μm (a-b, d-e) or 1 μm (c) . Linear mixed model (a, d-e) or negative binomial generalized linear model (c) followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Staining, Over Expression, Marker, Electron Microscopy, Imaging, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Syx13 protein level was increased upon rapamycin treatment in young fat body cells and this increase was partly reverted by S6K overexpression ( Lsp2GS>S6K CA ; rapamycin: p=0.0003, Lsp2GS>S6K CA induction: p=0.0138, interaction p=0.0086, n=5). b , Syx13 protein level was increased upon S6K repression ( Lsp2GS>S6K RNAi ; p=0.0226, n=5) in young fat body cells. Syx13 protein levels were measured by mass spectrometry-based proteomics. c , Knock-down of Syx13 expression ( Lsp2GS>Syx13 RNAi ) resulted in enlarged lysosomes in the fat body of young flies, depicted by lysotracker staining (p=0.0057, n=14). d , Overexpression of Syx13 ( Lsp2GS>Syx13 ) did not affect lysosomal enlargement (p=0.2894, n=12) in young fat bodies. e , Overexpression of Syx13 ( Lsp2GS>S6K CA ;Syx13 ) rescued the enlarged lysosomes of flies overexpressing S6K ( Lsp2GS>S6K CA ) treated with rapamycin ( Lsp2GS>S6K CA induction: p<0.0001, Lsp2GS>Syx13 induction: p<0.0001, interaction p=0.0429, n=12). f , Overexpression of Syx13 ( Lsp2GS>S6K CA ;Syx13 ) partially rescued the multilamellar lysosomes of S6K overexpressing ( Lsp2GS>S6K CA ) flies treated with rapamycin, depicted by electron microscopy ( Lsp2GS>S6K CA induction: p=0.0005, Lsp2GS>Syx13 induction: p=0.7989, interaction p=0.3068, n=5). Data are displayed as mean□±□s.e.m. (a, b, f) or displayed as Tukey box plot (c-e) . Each data point represents an average value per five fat bodies (a-b ) or per fat body (c-f) . Scale bar, 10 μm (c-e) or 1 μm (f) . Linear mixed model (a-e) or negative binomial generalized linear model (f) followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Over Expression, Mass Spectrometry, Expressing, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a , Cleaved Relish (Rel49, 49 kDa) in fat bodies of young (day 10), middle (day 30), and old (day 50) flies (age effect p=0.0034, n=4). b-c , Relish protein localisation ( b , n=9 in young and n=13 in old) and DptA transcript expression ( c , n=4) in fat bodies of young and old flies. d-f , S6K inhibition ( Lsp2GS>S6K RNAi ) suppressed the age-related increase in activated Rel49 ( d , n=5), accumulation of Relish in the nucleus ( e , n=14), and the increase in DptA expression ( f , n=3). g-h , Rapamycin treatment suppressed age-related Relish localisation (g) and the increase in DptA (h) . Overexpression of S6K ( Lsp2GS>S6K CA ) blocked the effect of rapamycin on Relish localisation ( g , rapamycin: p=0.0025, Lsp2GS>S6K CA induction: p<0.0001, interaction p=0.2345, n=14) and DptA expression ( h , n=4). i , Rapamycin treatment improved bacterial clearance in old flies infected with Ecc15 . This effect was blocked by S6K overexpression ( Lsp2GS>S6K CA ) (rapamycin: p=0.0185, Lsp2GS>S6K CA induction: p=0.0024, interaction p=0.0620, n=12). Data are displayed as mean□±□s.e.m. (a, c-d, f, h) or Tukey box plot (b, e, g, i) . Each data point represents an average value per fat body (b, e, g ), per five fat bodies (a, c, d, f, h) , or per three whole flies (i) . Scale bar, 10 μm. One-way ANOVA followed by Dunnett’s multiple comparison test (a); linear mixed model (b, e, g) or two-way ANOVA with log transformation (i) followed by Tukey’s multiple comparison test; two-sided Student’s t-test with (c, f, h) or without log-transformation (d) .

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Expressing, Inhibition, Over Expression, Infection, Transformation Assay

Journal: bioRxiv

Article Title: Inhibition of S6K lowers age-related inflammation and immunosenescence and increases lifespan through the endolysosomal system

doi: 10.1101/2022.08.25.505264

Figure Lengend Snippet: a-c , Rapamycin treatment suppressed the age-related nuclear localisation of Relish in old fat body cells. Dominant negative Rab7 ( a , rapamycin: p=0.0638, Lsp2GS>Rab7 DN induction: p<0.0001, interaction p=0.0160, n=14) and Rab5 ( b , rapamycin: p<0.0001, Lsp2GS>Rab5 DN induction: p<0.0001, interaction: p=0.0003, n=14) blocked the effect of rapamycin on the age-related nuclear localisation of Relish. c-d , Knockdown of Syx13 ( Lsp2GS>Syx13 RNAi ) blocked the effect of rapamycin on ( c ) Relish nuclear localisation (rapamycin: p=0.0012, Lsp2GS>Syx13 RNAi induction: p<0.0001, interaction p=0.2930, n=14) and ( d) bacterial clearance (rapamycin: p=0.0093, Lsp2GS>Syx13 RNAi induction: p=0.0057, interaction p=0.2226, n=8). e , Knockdown of Syx13 ( Lsp2GS>S6K RNAi ;Syx13 RNAi ) blocked the effect of S6K knockdown on Relish nuclear localisation ( Lsp2GS>S6K RNAi induction: p=0.0004, Lsp2GS>Syx13 RNAi induction: p<0.0001, interaction: p=0.0088, n=14). f-g , Overexpression of Syx13 ( Lsp2GS>S6K CA ;Syx13 ) rescued the effect of S6K activation on Relish localisation ( f , Lsp2GS>S6K CA induction: p<0.0001, Lsp2GS>Syx13 induction: p<0.0001, interaction p<0.0001, n=14) and bacterial clearance ( g , Lsp2GS>S6K CA induction: p=0.0644, Lsp2GS>Syx13 induction: p=0.7042, interaction: p=0.0429, n=10) in old flies treated with rapamycin. h-i , Middle-age-onset repression of Relish using Lsp2GS>Relish RNAi improved bacterial clearance ( h , n=8 in 0 h.p.i. group and n=12 in 12 h.p.i. groups) and extended lifespan ( i , p=0.0091, n=200). Data are displayed as Tukey box plot. Each data point represents an average value per fat body (a-c, e-f) or per three whole flies (d, g-h) . Scale bar, 10 μm. Linear mixed model (a-c, e-f) , two-way ANOVA with log transformation (d, g) followed by Tukey’s multiple comparison test; two-sided Student’s t-test (h) ; log-rank test (i) .

Article Snippet: Membranes were blocked by Intercept TBS Blocking Buffer (LI-COR, #927-60001) for 1 h and probed with the following primary antibodies diluted in Intercept T20 TBS Antibody Diluent (LI-COR, #927-65001): anti-S6K (1:1,000), anti-pS6K T398 (Cell Signaling Technology, #9209, 1:1,000), anti-Relish (Developmental Studies Hybridoma Bank, #21F3, 1:100), anti-tubulin (Sigma, #T9026,1:5,000), anti-puromycin (Sigma, #MABE343, 1:1,000), anti-Stx12 (Synaptic Systems, #110 132, 1:1000).

Techniques: Dominant Negative Mutation, Over Expression, Activation Assay, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 2. Inhibition of UVB-induced phosphorylation of Akt and p70 S6-K by pretreatment of cells with EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with EGCG or theaflavins for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels were estimated by immunoblotting.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Phospho-proteomics, Concentration Assay, Irradiation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 3. Inhibition of UVB-induced activation of Akt and p70 S6-K by pretreatment of cells with EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with EGCG or theaflavins for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunopre- cipitated using anti-Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. * and **, significant difference from the UVB control at p 0.05 and p 0.01, respectively. B, the cells were lysed, and p70 S6-K was immuno- precipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. *, signifi- cant difference from the UVB control at p 0.05.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Concentration Assay, Irradiation, Cell Culture, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 4. Inhibition of UVB-induced phosphorylation of Akt and p70 S6-K by pretreatment of cells with U0126, SB202190, LY294002, or rapamycin. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with U0126, SB202190, LY294002, or rapamycin for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels were estimated by immunoblotting.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Phospho-proteomics, Concentration Assay, Irradiation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 5. Inhibition of UVB-induced activation of Akt and p70 S6-K by pretreatment of cells with U0126, SB202190, LY294002 or rapamycin. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with U0126, SB202190, LY294002, or rapa- mycin for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunoprecipitated using a Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least two independent experiments. *, significant difference from the UVB control at p 0.01. B, the cells were lysed, and p70 S6-K was immunoprecipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. *, significant difference from the UVB con- trol at p 0.01.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Concentration Assay, Irradiation, Cell Culture, Immunoprecipitation, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 6. UVB-induced phosphorylation and activation of p70 S6-K in cells expressing dominant negative mutant of JNK1. A, JB6 Cl 41 cells and its stable transfectants, Cl 41 CMV-neo, and Cl 41 dominant negative mutant of JNK1 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then irradiated with UVB (4 kJ/m2) and cultured for 30 min. The cells were lysed, and the activity of JNKs was determined as described under “Experimental Procedures.” B, the transfectant cells were treated as described above and lysed. The levels of phosphoryla- tion of p70 S6-K were estimated by immunoblotting. C, JB6 Cl 41 and dominant negative mutant of JNK1 cells were treated as described above and lysed. p70 S6-K was immunoprecipitated from the lysates using a p70 S6-K antibody. The activities of p70 S6-K were assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least two independent experiments.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Phospho-proteomics, Activation Assay, Expressing, Dominant Negative Mutation, Irradiation, Cell Culture, Activity Assay, Transfection, Western Blot, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 7. Direct inhibition of UVB-induced p70 S6-K activation by EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunoprecipitated using a Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP with different concentrations of EGCG or theaflavins. Each bar indicates the mean S.E. of at least three independent experi- ments. B, the cells were lysed, and p70 S6-K was immunoprecipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP with different concentrations of EGCG or theaflavins. Each bar indicates the mean S.E. of at least three independent experiments. *, significant difference from the UVB control at p 0.05.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Irradiation, Cell Culture, Immunoprecipitation, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 8. Effect of tea polyphenols on PI3K-dependent pathway induced by UVB irradiation. Exposure to UVB results in activation of the PI3K pathway. EGCG and theaflavins inhibit UVB-induced ac- tivation and phosphorylation of PI3K and its downstream effectors, Akt (Thr308/Ser473) and p70 S6-K (Thr389 and Thr421/Ser424). The tea poly- phenols did not affect phosphorylation of Akt (Thr308) by PDK-1. UVB- induced p70 S6-K activation is also blocked by EGCG or theaflavins. The Erk kinase pathway, critical for UVB-induced activities of Akt and p70 S6-K, is also inhibited by tea polyphenols. 1, activation; , inhi- bition; tea bags, tea polyphenols.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Irradiation, Activation Assay, Phospho-proteomics

Journal: Endocrinology

Article Title: AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma.

doi: 10.1210/en.2005-0172

Figure Lengend Snippet: FIG. 6. A, Expression of a dominant-negative AKT (DNAKT) in cells reduces downstream targets of AKT. Infection of a primary thyroid cell line using either a control adenovirus containing -gal or an HA-tagged DNAKT was performed at an MOI of 100. Western blot of the cell lysates confirms the presence of the HA epitope only in the DNAKT infected cells. The level of phosphorylated mammalian target of rapamycin (phos-mTOR), and phosphorylated p70 S6K (phos-p70 S6K) downstream targets of AKT, is reduced in the DNAKT-infected cells. Protein disulfide isomerase (PDI) was used as a control for loading. Representative data from two independent experiments are shown. B, Inhibition of AKT signaling impairs primary thyroid cell motility. Cell motility assay of the adenovirus-infected cell lines, com- pared with control, show a 35% reduction of cell motility in cells expressing the DNAKT. Data are presented as the mean SEM of two independent experiments performed in triplicate (n 6).

Article Snippet: Antibodies used according to the manufacturers’ manuals include phospho-AKT (Ser473) no. 9271 (Cell Signaling Technologies, Beverly, MA), AKT no. 9272 (Cell Signaling Technologies) at 1:1000 dilution, hemagglutinin (HA) sc-7392 (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200, phospho-mammalian target of rapamycin (mTOR) (Ser2448) no. 2971 (Cell Signaling Technologies) at 1:1000, and phosphop70 S6K (Thr421/Ser424) no. 9204 (Cell Signaling Technologies) at 1:1000 dilution.

Techniques: Expressing, Dominant Negative Mutation, Infection, Control, Western Blot, Inhibition, Motility Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 2. Inhibition of UVB-induced phosphorylation of Akt and p70 S6-K by pretreatment of cells with EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with EGCG or theaflavins for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels were estimated by immunoblotting.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Phospho-proteomics, Concentration Assay, Irradiation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 3. Inhibition of UVB-induced activation of Akt and p70 S6-K by pretreatment of cells with EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with EGCG or theaflavins for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunopre- cipitated using anti-Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. * and **, significant difference from the UVB control at p 0.05 and p 0.01, respectively. B, the cells were lysed, and p70 S6-K was immuno- precipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. *, signifi- cant difference from the UVB control at p 0.05.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Concentration Assay, Irradiation, Cell Culture, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 4. Inhibition of UVB-induced phosphorylation of Akt and p70 S6-K by pretreatment of cells with U0126, SB202190, LY294002, or rapamycin. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with U0126, SB202190, LY294002, or rapamycin for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels were estimated by immunoblotting.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Phospho-proteomics, Concentration Assay, Irradiation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 5. Inhibition of UVB-induced activation of Akt and p70 S6-K by pretreatment of cells with U0126, SB202190, LY294002 or rapamycin. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with U0126, SB202190, LY294002, or rapa- mycin for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunoprecipitated using a Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least two independent experiments. *, significant difference from the UVB control at p 0.01. B, the cells were lysed, and p70 S6-K was immunoprecipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. *, significant difference from the UVB con- trol at p 0.01.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Concentration Assay, Irradiation, Cell Culture, Immunoprecipitation, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 6. UVB-induced phosphorylation and activation of p70 S6-K in cells expressing dominant negative mutant of JNK1. A, JB6 Cl 41 cells and its stable transfectants, Cl 41 CMV-neo, and Cl 41 dominant negative mutant of JNK1 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then irradiated with UVB (4 kJ/m2) and cultured for 30 min. The cells were lysed, and the activity of JNKs was determined as described under “Experimental Procedures.” B, the transfectant cells were treated as described above and lysed. The levels of phosphoryla- tion of p70 S6-K were estimated by immunoblotting. C, JB6 Cl 41 and dominant negative mutant of JNK1 cells were treated as described above and lysed. p70 S6-K was immunoprecipitated from the lysates using a p70 S6-K antibody. The activities of p70 S6-K were assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least two independent experiments.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Phospho-proteomics, Activation Assay, Expressing, Dominant Negative Mutation, Irradiation, Cell Culture, Activity Assay, Transfection, Western Blot, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 7. Direct inhibition of UVB-induced p70 S6-K activation by EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunoprecipitated using a Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP with different concentrations of EGCG or theaflavins. Each bar indicates the mean S.E. of at least three independent experi- ments. B, the cells were lysed, and p70 S6-K was immunoprecipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP with different concentrations of EGCG or theaflavins. Each bar indicates the mean S.E. of at least three independent experiments. *, significant difference from the UVB control at p 0.05.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Irradiation, Cell Culture, Immunoprecipitation, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 8. Effect of tea polyphenols on PI3K-dependent pathway induced by UVB irradiation. Exposure to UVB results in activation of the PI3K pathway. EGCG and theaflavins inhibit UVB-induced ac- tivation and phosphorylation of PI3K and its downstream effectors, Akt (Thr308/Ser473) and p70 S6-K (Thr389 and Thr421/Ser424). The tea poly- phenols did not affect phosphorylation of Akt (Thr308) by PDK-1. UVB- induced p70 S6-K activation is also blocked by EGCG or theaflavins. The Erk kinase pathway, critical for UVB-induced activities of Akt and p70 S6-K, is also inhibited by tea polyphenols. 1, activation; , inhi- bition; tea bags, tea polyphenols.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Irradiation, Activation Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: mTORC1 phosphorylates LARP6 to stimulate type I collagen expression

doi: 10.1038/srep41173

Figure Lengend Snippet: ( a ) LARP6 phosphorylation changes in activation of HSCs. Primary rat HSCs were transduced by adenovirus expressing wt LARP6 on day 2 or on day 4 and analyzed by 2DGE on day 3 or on day 5 (CON, panels 1 and 2). The same cells were also treated with rapamycin on day 2 and day 4 and analyzed on day 3 and day 5 (panels 3 and 4). Panels 5 and 6; cells transduced with S348A/S409A mutant. ( b ) Increased AKT and mTORC1 signaling during HSCs activation. HSCs at day 3 and day 5 in culture were analyzed for expression of phospho-AKT and phospho-S6K. p-AKT: phopho-AKT. AKT: total AKT. p-S6K: phospho-S6K. S6K: total S6K. ACT: β-actin, loading control. ( c ) Dominant negative effect of S348A/S409A in HSCs. HSCs were transduced on day 3 with wt LARP6 and S348A/S409A mutant and on day 5 cellular (CELL) and medium (MED) level of COL1A1 was analyzed by Western blot. Loading controls: β-actin (ACT) and fibronectin (FIB). HA-LARP6, expression of the transduced proteins. ( d ) Rapamycin reduces expression of collagen by HSCs. HSCs cultured for 5 days were treated with or without RAPA for 2 h and COL1A1 polypeptide was analyzed intracellularly (CELL) or in the medium (MED).

Article Snippet: Antibodies used were: anti-LARP6 antibody from Abnova (H00055323-B01P), anti-HA antibody from Sigma-Aldrich (H9658), anti-phospho AKT (S473), anti-pan AKT, anti-phospho S6K (T389), and anti-S6K antibodies from Cell Signaling Technology (12694, 4691, 9205, and 2708, respectively), anti-collagen α1(I) antibody from Rockland (600-401-103), anti-collagen α2(I) antibody from Santa Cruz Biotechnology (sc-8786), anti-calnexin antibody, human anti-fibronectin antibody and anti-STRAP antibody from BD Transduction Laboratories (610523, 610077, and 611346, respectively), rat anti-fibronectin from Millipore (AB1954), anti-β-actin antibody from Abnova (ab8227), anti-Sec61β antibody from Thermo Scientific (PA3-015) and anti-raptor antibody from Bethyl Laboratories (A300-553A-M).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Transduction, Mutagenesis, Control, Dominant Negative Mutation, Western Blot, Cell Culture