trb3  (Millipore)


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    Name:
    Signal Transduction
    Description:
    This text explains how cells respond to external cues hormones cytokines neurotransmitters adhesion molecules extracellular matrix etc and shows how these inputs are integrated and coordinated The first half explains the formation and action of second messengers particularly cyclic nucleotides and calcium and the mediation of signal pathways by GTP binding proteins Later it deals with the formation of complex signaling cascades employed by cytokines and adhesion molecules starting at the membrane and ending in the nucleus to regulate gene transcription It ends with a description at the molecular level of how signaling proteins interact with their environment and with each other through their structural domains
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    s2066
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    Structured Review

    Millipore trb3
    Signal Transduction
    This text explains how cells respond to external cues hormones cytokines neurotransmitters adhesion molecules extracellular matrix etc and shows how these inputs are integrated and coordinated The first half explains the formation and action of second messengers particularly cyclic nucleotides and calcium and the mediation of signal pathways by GTP binding proteins Later it deals with the formation of complex signaling cascades employed by cytokines and adhesion molecules starting at the membrane and ending in the nucleus to regulate gene transcription It ends with a description at the molecular level of how signaling proteins interact with their environment and with each other through their structural domains
    https://www.bioz.com/result/trb3/product/Millipore
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trb3 - by Bioz Stars, 2020-08
    94/100 stars

    Images

    1) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    2) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    3) Product Images from "Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney"

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013070811

    Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.
    Figure Legend Snippet: Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.

    Techniques Used: Expressing

    Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.
    Figure Legend Snippet: Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.

    Techniques Used: Knock-Out, Southern Blot, Mouse Assay

    In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.
    Figure Legend Snippet: In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.

    Techniques Used: Mouse Assay, Staining

    TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P
    Figure Legend Snippet: TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P

    Techniques Used: Expressing, Transfection

    Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P
    Figure Legend Snippet: Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P
    Figure Legend Snippet: Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P
    Figure Legend Snippet: Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.
    Figure Legend Snippet: TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.

    Techniques Used: Binding Assay, Transformation Assay, Transfection, Immunoprecipitation, Mouse Assay, Western Blot

    4) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    5) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    6) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    7) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    8) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    9) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    10) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    11) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    12) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    13) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    14) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    15) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    16) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    17) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    18) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    19) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    20) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    21) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    22) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    23) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    24) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    25) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    26) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    27) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    28) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    29) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    30) Product Images from "Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney"

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013070811

    Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.
    Figure Legend Snippet: Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.

    Techniques Used: Expressing

    Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.
    Figure Legend Snippet: Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.

    Techniques Used: Knock-Out, Southern Blot, Mouse Assay

    In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.
    Figure Legend Snippet: In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.

    Techniques Used: Mouse Assay, Staining

    TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P
    Figure Legend Snippet: TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P

    Techniques Used: Expressing, Transfection

    Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P
    Figure Legend Snippet: Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P
    Figure Legend Snippet: Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P
    Figure Legend Snippet: Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.
    Figure Legend Snippet: TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.

    Techniques Used: Binding Assay, Transformation Assay, Transfection, Immunoprecipitation, Mouse Assay, Western Blot

    31) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    32) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    33) Product Images from "Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle"

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2017.09.134

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P
    Figure Legend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    34) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    35) Product Images from "TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1"

    Article Title: TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00236.2010

    ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P
    Figure Legend Snippet: ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H 2 O 2 ( A ) and 24 h with palmitate (Palm) or BSA control ( B ). CHOP expression was assessed by real-time PCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.
    Figure Legend Snippet: High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

    Techniques Used: Expressing, Incubation

    Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.
    Figure Legend Snippet: Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.

    Techniques Used: Activation Assay, Expressing

    TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P
    Figure Legend Snippet: TRB3 expression increases in diabetic mouse kidneys. A : C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice ( n = 5 mice per group). B : TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice ( n = 5 mice per group). * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P
    Figure Legend Snippet: Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H 2 O 2 ( A ) and 3 h with xanthine and xanthine oxidase ( B ). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P
    Figure Legend Snippet: Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A : real-time PCR studies demonstrated that FFA induce TRB3 expression. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P
    Figure Legend Snippet: A : TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. * P

    Techniques Used: Expressing, Transfection

    36) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    37) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    38) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    39) Product Images from "Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts"

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    Journal: Diabetes

    doi: 10.2337/db17-0219

    Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Effects of TRB3 manipulation on Akt function in WT and MT-TG cardiomyocytes. The efficacy of Ad-shRNA interference of TRB3 expression in WT adult cardiomyocytes and Ad-TRB3–mediated TRB3 and Ad-Akt1–mediated Akt1 overexpression in MT-TG adult cardiomyocytes were optimized in a pilot study ( Supplementary Fig. 1 ). Then the effects TRB3 knockdown ( A and B ) or overexpression with or without further forced overexpression of Akt1 ( H and I ) on the phosphorylation of total Akt2 ( A , C , H , and J ), GSK-3β ( A , D , H , and K ), and GS ( A , E , H , and L ) and expression of HK II ( A , F , H , and M ) and PGC-1α ( A , G , H , and N ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to control treatment. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: shRNA, Expressing, Over Expression, Pyrolysis Gas Chromatography, Western Blot

    Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Supplementation with Zn preserves cardiac Akt signaling and prevents DCM in db/db type 2 diabetic mice. db/db and littermate WT control mice at age of 10–12 weeks were fed an HZ or NZ diet for 3 months. Then, these mice were sacrificed for collecting heart tissues following cardiac function assay by echo ( Table 1 ). Cardiac MT expression ( A and B ), the phosphorylation of total Akt ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ), and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT control (WT-NZ). n ≥ 7. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Mouse Assay, Functional Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: Adenovirus-mediated TRB3 overexpression abolishes MT’s preservation of cardiac Akt signaling and prevention of cardiac damage in STZ-induced type 1 diabetes. The efficacy of the intramyocardial gene delivery was validated in 2-month-old MT-TG mice in a pilot study ( Supplementary Fig. 3 ). Two months after diabetes onset, Ad-TRB3 or Ad-GFP control was delivered by myocardial injection. One month after intramyocardial injection, these mice were sacrificed, and cardiac tissues were collected following cardiac function examination by echo ( Table 1 ). Cardiac oxidative stress was detected by a TBARS assay ( A ), and the phosphorylation of total Akt ( B and C ), Akt2 ( B and D ), and GSK-3β ( B and E ) and the expression of TRB3 ( B and F ), CTGF ( B and G ), TGF-β1 ( B and H ), PAI-1 ( B and I ), and TNF-α ( B and J ) were detected by Western blot. GAPDH was used for loading control. Results were normalized to the WT-Ctrl. The cardiac collagen accumulation was detected by Sirius Red staining ( K ). n = 7. Scale bars: 50 μM. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Over Expression, Preserving, Mouse Assay, Injection, TBARS Assay, Expressing, Western Blot, Staining

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P
    Figure Legend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Techniques Used: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    40) Product Images from "Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney"

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013070811

    Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.
    Figure Legend Snippet: Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.

    Techniques Used: Expressing

    Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.
    Figure Legend Snippet: Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.

    Techniques Used: Knock-Out, Southern Blot, Mouse Assay

    In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.
    Figure Legend Snippet: In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.

    Techniques Used: Mouse Assay, Staining

    TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P
    Figure Legend Snippet: TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P

    Techniques Used: Expressing, Transfection

    Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P
    Figure Legend Snippet: Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P

    Techniques Used: Knock-Out, Mouse Assay, Western Blot

    Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P
    Figure Legend Snippet: Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P
    Figure Legend Snippet: Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.
    Figure Legend Snippet: TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.

    Techniques Used: Binding Assay, Transformation Assay, Transfection, Immunoprecipitation, Mouse Assay, Western Blot

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    Article Snippet: .. Treatment with signal transduction inhibitors HEK293ar cells were treated with the ERK inhibitor PD98059 (0-50 μmol/l; Sigma, USA), the PI3K inhibitor LY294002 (0-50 μmol/l; Sigma, USA), or the DMSO vehicle as a control for 2 h under adhesion conditions. .. The treated cells were cultured under cell-detachment conditions for the indicated times.

    Article Title: Epidermal Growth Factor Receptor Signaling Is Partially Responsible for the Increased Matrix Metalloproteinase-1 Expression in Ocular Epithelial Cells after UVB Radiation
    Article Snippet: .. For inhibition of EGFR-mediated signal transduction, quiescent PECs were pretreated with PD153035 (0.1 to 5 μmol/L final, Calbiochem) for 1 hour before and for the specified times after irradiation or stimulated with either recombinant human EGF (Sigma) or HB-EGF (R & D Systems, Minneapolis, MN) to a final concentration of 100 ng/ml. .. PD153035 is an extremely potent and specific inhibitor of the tyrosine kinase activity of the EGFR and rapidly suppresses autophosphorylation at low nanomolar concentrations.

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    Article Title: Polysaccharide isolated from seeds of Plantago asiatica L. induces maturation of dendritic cells through MAPK and NF-κB pathway
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    Irradiation:

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    Blocking Assay:

    Article Title: Polysaccharide isolated from seeds of Plantago asiatica L. induces maturation of dendritic cells through MAPK and NF-κB pathway
    Article Snippet: .. To block NF-κB pathway signal transduction, the cells were pre-treated with 30 μM pyrrolidine dithiocarbamate (PDTC, Sigma-Aldrich, USA) for 40 min. .. Besides, DCs were pre-treated with 30 μM anti-TLR4/MD2 monoclonal antibody (eBioscience, USA) for 1 h to block toll-like receptor-4 (TLR-4) signal recognition.

    Concentration Assay:

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    Article Snippet: .. For inhibition of EGFR-mediated signal transduction, quiescent PECs were pretreated with PD153035 (0.1 to 5 μmol/L final, Calbiochem) for 1 hour before and for the specified times after irradiation or stimulated with either recombinant human EGF (Sigma) or HB-EGF (R & D Systems, Minneapolis, MN) to a final concentration of 100 ng/ml. .. PD153035 is an extremely potent and specific inhibitor of the tyrosine kinase activity of the EGFR and rapidly suppresses autophosphorylation at low nanomolar concentrations.

    Inhibition:

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    Article Snippet: .. For inhibition of EGFR-mediated signal transduction, quiescent PECs were pretreated with PD153035 (0.1 to 5 μmol/L final, Calbiochem) for 1 hour before and for the specified times after irradiation or stimulated with either recombinant human EGF (Sigma) or HB-EGF (R & D Systems, Minneapolis, MN) to a final concentration of 100 ng/ml. .. PD153035 is an extremely potent and specific inhibitor of the tyrosine kinase activity of the EGFR and rapidly suppresses autophosphorylation at low nanomolar concentrations.

    Western Blot:

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    Recombinant:

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    Millipore trb3
    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and <t>TRB3</t> ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P
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    MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Journal: Diabetes

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    doi: 10.2337/db17-0219

    Figure Lengend Snippet: MT prevents tBHP inhibition of Akt function in vitro. Adult cardiomyocytes from WT and MT-TG mice were directly exposed to tBHP (100 μmol/L) for 2 h or LPS (10 µg/mL) for 4 h and then stimulated with or without insulin (INS; 100 nmol/L) for 15 min. Cells were collected, and the phosphorylation of total Akt ( A and B ), Akt2 ( A , C , I , and J ), GSK-3β ( A , D , I , and K ), and GS ( A , E , I , and L ) and expression of HK II ( A , F , I , and M ), PGC-1α ( A , G , I , and N ), and TRB3 ( A , H , I , and O ) was detected by Western blot. Results were normalized to WT control. Three independent experiments were performed. Data shown in graphs represent the means ± SD. * P

    Article Snippet: TRB3 is an Akt-negative regulator that directly binds to and blocks Akt phosphorylation ( , , ).

    Techniques: Inhibition, In Vitro, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Journal: Diabetes

    Article Title: Metallothionein Preserves Akt2 Activity and Cardiac Function via Inhibiting TRB3 in Diabetic Hearts

    doi: 10.2337/db17-0219

    Figure Lengend Snippet: MT prevents diabetic downregulation of cardiac Akt functions along with inhibition of TRB3 in STZ-induced type 1 diabetes. At 3 months after diabetes onset, WT and MT-TG mice were sacrificed, and cardiac tissue was used to analyze the phosphorylation of total Akt ( A and B ), Akt1 ( A and C ), Akt2 ( A and D ), GSK-3β ( A and E ), and GS ( A and F ) and the expression of HK II ( A and G ), PGC-1α ( A and H ), and TRB3 ( A and I ) by Western blot. Results were normalized to the WT controls. GAPDH was used as loading control. n = 7 for each group. Data shown in graphs represent the means ± SD. * P

    Article Snippet: TRB3 is an Akt-negative regulator that directly binds to and blocks Akt phosphorylation ( , , ).

    Techniques: Inhibition, Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Western Blot

    Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: Proposed mechanisms of protective role of TRB3 in diabetic kidney disease. The diabetic milieu activates the ER stress pathway, including protein kinase RNA–like ER kinase (PERK). PERK phosphorylates eukaryotic translation initiation factor- α to inhibit protein translation with selective translation of ATF4, which induces expression of CHOP and drives TRB3 expression. We demonstrate that in kidney cells TRB3 binds to Rictor and mTORC2 to inhibit phosphorylation of pAKT Ser473 , which can reduce inflammatory gene expression, including IL-6, MCP-1, and Lcn2/Ngal. ROS, reactive oxygen species.

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Expressing

    Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: Strategy to generate TRB3 knockout mouse. (A) Representation of targeted disruption of the mouse TRB3 gene. (B) Southern blot of Bam Hl digested genomic DNA from TRB3 +/− −/− and +/+ mice.

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Knock-Out, Southern Blot, Mouse Assay

    In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: In diabetic mice, absence of TRB3 does not markedly alter glomerular histologic features. Morphometry was performed on PAS-stained kidney sections. (A) WT control (B) WT STZ, (C) TRB3 −/− control, and (D) TRB3 −/− STZ-treated mice. (E) PAS-positive matrix, (F) glomerular nuclei, and (G) glomerular area were assessed as described in Concise Methods. n =4 WT control, n =5 TRB3 −/− control, n =5 WT STZ, n =6 TRB3 −/− STZ. Bars represent SEM.

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Mouse Assay, Staining

    TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: TRB3 inhibits IL-6 expression in MCT cells. MCT cells were transfected with pcDNA3 or pcDNA3-HA-TRB3 and grown in normal-glucose (5.5 mM) and high-glucose (30.5 mM) conditions. Eighteen hours after transfection, supernatants were collected, and ELISAs detected (A) IL-6 expression pg/ml supernatant and (B) IL-6 pg/mg (protein). * P

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Expressing, Transfection

    Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: Knockout of TRB3 increases phosphorylation of AKT at Ser 473 and reduces the phosphorylation at Thr 308 in renal cortices of diabetic mice. (A) Western blotting of pAKT Ser473 , pAKT Thr308 , and total AKT in the experimental mice. Densitometry of (B) pAKT Ser473 /AKT and (C) pAKT Thr308 /AKT. * P

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Knock-Out, Mouse Assay, Western Blot

    Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: Diabetic TRB3 knockout mice have higher renal inflammatory gene expression and evidence of ER stress than WT controls. Real-time PCR on kidney cortices comparing diabetic with nondiabetic controls revealed the following: (A) increased TRB3 expression in the diabetic WT mice compared with nondiabetic WT mice and higher (B) MCP-1, (C) IL-6, (D) Lcn2/Ngal, and (E) fibronectin expression in diabetic TRB3 −/− mice than in WT diabetic mice. (F) There was no difference in TGF- β mRNA expression in the diabetic WT and TRB3 −/− mice. (G–I) There was higher renal cortical expression of the ER stress markers (GRP78/BiP and ATF4) and a trend for higher CHOP in the diabetic TRB3 −/− mice. In both genotypes, there was higher excretion of (J) the early-response cytokine IL-6 in 24-hour urine collected 4 weeks after STZ administration and (K) MCP-1 and (L) Lcn2/Ngal in urine collected at 8 weeks after diabetes induction. * P

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Knock-Out, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: Absence of TRB3 augments ER stress in the glomeruli of diabetic mice. Real-time PCR on glomerular preparations comparing diabetic with nondiabetic controls revealed expression of (A) TRB3, (B) GRP78/BiP, (C) Lcn2/Ngal, and (D) CHOP mRNA. * P

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Tribbles Homolog 3 Attenuates Mammalian Target of Rapamycin Complex-2 Signaling and Inflammation in the Diabetic Kidney

    doi: 10.1681/ASN.2013070811

    Figure Lengend Snippet: TRB3 may inhibit phosphorylation of AKT Ser473 by binding to Rictor and mTORC2. (A) Fully differentiated transformed podocytes were transfected with pcDNA3-HA-TRB3 (HA-TRB3), which consistently reduced phosphorylation of AKT at Ser 473 (pAKT Ser473 ) (B) Densitometry results of transfection of HA-TRB3 or pcDNA3 in transformed podocytes (study performed three times). (C) MCT cells were transfected with HA-TRB3 or pcDNA3, and the TRB3-containing complexes were immunoprecipitated with anti-HA beads. TRB3 binds to Rictor and mTOR, but not directly to AKT or pAKT. This study is representative of studies performed three times. (D) Primary podocytes were grown from WT and TRB3 −/− mice. Western blotting revealed higher phosphorylation of pAKT Ser473 in primary podocytes grown from TRB3 −/− mice (study is representative of Western blotting performed twice). (E) MCT cells were transfected with pcDNA3 or HA-TRB3, and Western blotting for targets of mTORC2 and AKT was assessed. Bars represent SEM.

    Article Snippet: Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver.

    Techniques: Binding Assay, Transformation Assay, Transfection, Immunoprecipitation, Mouse Assay, Western Blot

    Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Journal: Biochemical and biophysical research communications

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    doi: 10.1016/j.bbrc.2017.09.134

    Figure Lengend Snippet: Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle GAS muscles were collected from WT and TRB3TG mice. (A–D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E–G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 µmol/g body weight with puromycin dissolved in 100 µl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Article Snippet: Genetic deletion of Trb3, the mammalian Drosophila tribbles homolog, displays normal hepatic insulin signaling and glucose homeostasis.

    Techniques: Over Expression, Mouse Assay, Western Blot, Injection, Staining

    Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Journal: Biochemical and biophysical research communications

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    doi: 10.1016/j.bbrc.2017.09.134

    Figure Lengend Snippet: Effects of TRB3 on muscle mass and function in mice (A–B) Ten- to eleven-weekold female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E–F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-µm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: ×400; scale bar: 50 µm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5–6 per group). * P

    Article Snippet: Genetic deletion of Trb3, the mammalian Drosophila tribbles homolog, displays normal hepatic insulin signaling and glucose homeostasis.

    Techniques: Mouse Assay, Transgenic Assay, Expressing, Staining

    Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Journal: Biochemical and biophysical research communications

    Article Title: Tribbles 3 Regulates Protein Turnover in Mouse Skeletal Muscle

    doi: 10.1016/j.bbrc.2017.09.134

    Figure Lengend Snippet: Effects of TRB3 knockout on protein synthesis and muscle mass Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A–D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F–G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H–I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5–6 per group). * P

    Article Snippet: Genetic deletion of Trb3, the mammalian Drosophila tribbles homolog, displays normal hepatic insulin signaling and glucose homeostasis.

    Techniques: Knock-Out, Mouse Assay, Western Blot