phospho akt thr308  (Cell Signaling Technology Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Phospho Akt Thr308 Antibody
    Description:
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19 Description Cell Signaling Technology antibody formulated in PBS no BSA no glycerol and quality controlled for use in ELISA and other drug discovery applications This is a sample antibody and intended for use by drug discovery scientists Please inquire for bulk pricing of this antibody
    Catalog Number:
    5110
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr308 of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc phospho akt thr308
    LS-induced upregulation of KCa3.1 and KCa2.3 expression is mediated by CaMKK-dependent <t>Akt</t> activation. A and B : HCAECs were exposed to LS for 24 h in the absence or presence of compound C (an AMPK inhibitor), KN-62 (a CaMK inhibitor), or Akt inhibitor IV. Compound C tended to reduce LS-induced upregulation of KCa3.1 mRNA expression ( A ) but did not change LS induction of KCa2.3 expression ( B ) ( n = 9). KN-62 had no effect on the upregulation of KCa3.1 and KCa2.3 mRNA expression ( n = 7–8). Akt inhibition completely blocked LS-induced upregulation of both KCa3.1 and KCa2.3 expression. C : LS-induced increase in KCa2.3 and KCa3.1 protein expression levels was suppressed by Akt inhibition. Top : a representative image of immunoblots. Bottom : the summaries of densitometric analysis of KCa3.1 and KCa2.3 signals ( n = 3). D : Akt inhibitor IV abolished the phosphorylation of Akt at Ser473 in HCAECs exposed to 60-min LS ( top ). Levels of phospho-Akt (Ser473) were suppressed after inhibiting CaMKK with STO-609 in ECs exposed to 15 min of LS ( bottom ). Phosphorylation of Akt at <t>Thr308</t> was not observed in ECs under ST or LS condition. #Signficant difference vs. LS ( P
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19 Description Cell Signaling Technology antibody formulated in PBS no BSA no glycerol and quality controlled for use in ELISA and other drug discovery applications This is a sample antibody and intended for use by drug discovery scientists Please inquire for bulk pricing of this antibody
    https://www.bioz.com/result/phospho akt thr308/product/Cell Signaling Technology Inc
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    phospho akt thr308 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade"

    Article Title: Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00642.2012

    LS-induced upregulation of KCa3.1 and KCa2.3 expression is mediated by CaMKK-dependent Akt activation. A and B : HCAECs were exposed to LS for 24 h in the absence or presence of compound C (an AMPK inhibitor), KN-62 (a CaMK inhibitor), or Akt inhibitor IV. Compound C tended to reduce LS-induced upregulation of KCa3.1 mRNA expression ( A ) but did not change LS induction of KCa2.3 expression ( B ) ( n = 9). KN-62 had no effect on the upregulation of KCa3.1 and KCa2.3 mRNA expression ( n = 7–8). Akt inhibition completely blocked LS-induced upregulation of both KCa3.1 and KCa2.3 expression. C : LS-induced increase in KCa2.3 and KCa3.1 protein expression levels was suppressed by Akt inhibition. Top : a representative image of immunoblots. Bottom : the summaries of densitometric analysis of KCa3.1 and KCa2.3 signals ( n = 3). D : Akt inhibitor IV abolished the phosphorylation of Akt at Ser473 in HCAECs exposed to 60-min LS ( top ). Levels of phospho-Akt (Ser473) were suppressed after inhibiting CaMKK with STO-609 in ECs exposed to 15 min of LS ( bottom ). Phosphorylation of Akt at Thr308 was not observed in ECs under ST or LS condition. #Signficant difference vs. LS ( P
    Figure Legend Snippet: LS-induced upregulation of KCa3.1 and KCa2.3 expression is mediated by CaMKK-dependent Akt activation. A and B : HCAECs were exposed to LS for 24 h in the absence or presence of compound C (an AMPK inhibitor), KN-62 (a CaMK inhibitor), or Akt inhibitor IV. Compound C tended to reduce LS-induced upregulation of KCa3.1 mRNA expression ( A ) but did not change LS induction of KCa2.3 expression ( B ) ( n = 9). KN-62 had no effect on the upregulation of KCa3.1 and KCa2.3 mRNA expression ( n = 7–8). Akt inhibition completely blocked LS-induced upregulation of both KCa3.1 and KCa2.3 expression. C : LS-induced increase in KCa2.3 and KCa3.1 protein expression levels was suppressed by Akt inhibition. Top : a representative image of immunoblots. Bottom : the summaries of densitometric analysis of KCa3.1 and KCa2.3 signals ( n = 3). D : Akt inhibitor IV abolished the phosphorylation of Akt at Ser473 in HCAECs exposed to 60-min LS ( top ). Levels of phospho-Akt (Ser473) were suppressed after inhibiting CaMKK with STO-609 in ECs exposed to 15 min of LS ( bottom ). Phosphorylation of Akt at Thr308 was not observed in ECs under ST or LS condition. #Signficant difference vs. LS ( P

    Techniques Used: Expressing, Activation Assay, Inhibition, Western Blot

    2) Product Images from "Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells"

    Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071490

    Increased serum phosphorylates retinoblastoma, Akt Ser473 , and AKT Thr308 and decreases p27 KIP1 in confluent PASMC, but not in confluent PAEC. A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.
    Figure Legend Snippet: Increased serum phosphorylates retinoblastoma, Akt Ser473 , and AKT Thr308 and decreases p27 KIP1 in confluent PASMC, but not in confluent PAEC. A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

    Techniques Used: Western Blot, Infection, BrdU Incorporation Assay

    3) Product Images from "Diabetes Worsens Skeletal Muscle Mitochondrial Function, Oxidative Stress, and Apoptosis After Lower-Limb Ischemia-Reperfusion: Implication of the RISK and SAFE Pathways?"

    Article Title: Diabetes Worsens Skeletal Muscle Mitochondrial Function, Oxidative Stress, and Apoptosis After Lower-Limb Ischemia-Reperfusion: Implication of the RISK and SAFE Pathways?

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00579

    Protein levels of key components of the endogenous protective RISK and SAFE pathways. Quantification of the levels of effector proteins from the RISK pathway [Akt, phospho-Akt/Total Akt Thr308 (A) , phospho-Akt/Total Akt Ser473 (B) ] and the SAFE pathway [STAT3, phospho-STAT3/Total STAT3 tyr705 (C) ] in gastrocnemius muscle from sham-operated, vehicle-treated (nCON) or streptozotocin-treated rats (dCON), and after ischemia reperfusion in vehicle-treated (nIR) and streptozotocin-treated rats (dIR). GAPDH was used as an internal control for all proteins. Data are expressed as the mean ± SEM of the intensity of the bands reported to the intensity of the internal control. * p
    Figure Legend Snippet: Protein levels of key components of the endogenous protective RISK and SAFE pathways. Quantification of the levels of effector proteins from the RISK pathway [Akt, phospho-Akt/Total Akt Thr308 (A) , phospho-Akt/Total Akt Ser473 (B) ] and the SAFE pathway [STAT3, phospho-STAT3/Total STAT3 tyr705 (C) ] in gastrocnemius muscle from sham-operated, vehicle-treated (nCON) or streptozotocin-treated rats (dCON), and after ischemia reperfusion in vehicle-treated (nIR) and streptozotocin-treated rats (dIR). GAPDH was used as an internal control for all proteins. Data are expressed as the mean ± SEM of the intensity of the bands reported to the intensity of the internal control. * p

    Techniques Used:

    4) Product Images from "Diabetes Worsens Skeletal Muscle Mitochondrial Function, Oxidative Stress, and Apoptosis After Lower-Limb Ischemia-Reperfusion: Implication of the RISK and SAFE Pathways?"

    Article Title: Diabetes Worsens Skeletal Muscle Mitochondrial Function, Oxidative Stress, and Apoptosis After Lower-Limb Ischemia-Reperfusion: Implication of the RISK and SAFE Pathways?

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00579

    Protein levels of key components of the endogenous protective RISK and SAFE pathways. Quantification of the levels of effector proteins from the RISK pathway [Akt, phospho-Akt/Total Akt Thr308 (A) , phospho-Akt/Total Akt Ser473 (B) ] and the SAFE pathway [STAT3, phospho-STAT3/Total STAT3 tyr705 (C) ] in gastrocnemius muscle from sham-operated, vehicle-treated (nCON) or streptozotocin-treated rats (dCON), and after ischemia reperfusion in vehicle-treated (nIR) and streptozotocin-treated rats (dIR). GAPDH was used as an internal control for all proteins. Data are expressed as the mean ± SEM of the intensity of the bands reported to the intensity of the internal control. * p
    Figure Legend Snippet: Protein levels of key components of the endogenous protective RISK and SAFE pathways. Quantification of the levels of effector proteins from the RISK pathway [Akt, phospho-Akt/Total Akt Thr308 (A) , phospho-Akt/Total Akt Ser473 (B) ] and the SAFE pathway [STAT3, phospho-STAT3/Total STAT3 tyr705 (C) ] in gastrocnemius muscle from sham-operated, vehicle-treated (nCON) or streptozotocin-treated rats (dCON), and after ischemia reperfusion in vehicle-treated (nIR) and streptozotocin-treated rats (dIR). GAPDH was used as an internal control for all proteins. Data are expressed as the mean ± SEM of the intensity of the bands reported to the intensity of the internal control. * p

    Techniques Used:

    5) Product Images from "Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway ▿"

    Article Title: Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00797-10

    Akt inhibition blocks the effect of insulin on glucose transport uptake but not lipolysis. (A) 3T3L1 adipocytes were subjected to a glycerol release assay with increasing doses of isoproterenol in the absence or presence of insulin (1 nM) or Akt inhibitor (5 μM) for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. Immunoblots for phospho-Akt Thr308 and phospho-AS160/TBC1D4 using phospho-Akt substrate antibody were performed on cell lysates treated with the indicated conditions, including isoproterenol (2 nM), insulin (25 nM), and Akt inhibitor (20 μM). (B) 3T3-L1 adipocytes were used to generate an insulin dose-response curve of glycerol release (left) and fatty acid release (right) at a low concentration of isoproterenol (2 nM) in the presence and absence of Akt inhibitor (10 μM). Data are expressed as means ± SD from two experiments. (C) Simultaneous glycerol release and glucose uptake assays were performed on cells plated on the same day and cultured side by side with the indicated additions at the following concentrations: isoproterenol (2 nM), insulin (25 nM), and Akt inhibitor (20 μM). Data are expressed as means ± standard errors of the means from three experiments for glycerol release and means ± SD from two experiments for glucose uptake. *, P
    Figure Legend Snippet: Akt inhibition blocks the effect of insulin on glucose transport uptake but not lipolysis. (A) 3T3L1 adipocytes were subjected to a glycerol release assay with increasing doses of isoproterenol in the absence or presence of insulin (1 nM) or Akt inhibitor (5 μM) for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. Immunoblots for phospho-Akt Thr308 and phospho-AS160/TBC1D4 using phospho-Akt substrate antibody were performed on cell lysates treated with the indicated conditions, including isoproterenol (2 nM), insulin (25 nM), and Akt inhibitor (20 μM). (B) 3T3-L1 adipocytes were used to generate an insulin dose-response curve of glycerol release (left) and fatty acid release (right) at a low concentration of isoproterenol (2 nM) in the presence and absence of Akt inhibitor (10 μM). Data are expressed as means ± SD from two experiments. (C) Simultaneous glycerol release and glucose uptake assays were performed on cells plated on the same day and cultured side by side with the indicated additions at the following concentrations: isoproterenol (2 nM), insulin (25 nM), and Akt inhibitor (20 μM). Data are expressed as means ± standard errors of the means from three experiments for glycerol release and means ± SD from two experiments for glucose uptake. *, P

    Techniques Used: Inhibition, Glucose Assay, Western Blot, Concentration Assay, Cell Culture

    Differential effects of Akt inhibition at low and high concentrations of isoproterenol. (A) Floxed Akt2 fibroblasts stably expressing PPARγ were infected with either Adeno-GFP or Adeno-Cre and then differentiated into adipocytes. These cells were serum starved and treated with 1 nM insulin and analyzed by immunoblotting using Licor Odyssey for the excision of Akt2 and the loss of phospho-Akt Ser473 signal (left panel; representative of two experiments). The quantification of immunoblot analysis of phospho-Akt Ser473 of Akt2 lox/lox adipocytes infected with Ad-GFP or Ad-Cre is shown (middle panel). A glycerol release assay (right panel) was performed with increasing doses of isoproterenol in the presence and absence of 1 nM insulin for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. (B) RNA inteference-mediated reduction in Akt2 does not affect insulin inhibition of glycerol release. 3T3-L1 preadipocytes were infected with an shRNA lentiviral construct that targets Akt2 and sorted for the low and high expression of GFP. Adipocytes were treated with the indicated concentrations of insulin and subjected to the immunoblot analysis of Akt2 and phospho-Akt Thr308, confirming the efficiency of knockdown (left). Highly expressing cells were differentiated into adipocytes, and a glycerol release assay was performed using 2 nM isoproterenol with increasing doses of insulin (upper right). Data are expressed as means ± SD from two experiments performed in duplicate. A glucose uptake assay also was performed on differentiated control, highly, and lowly expressing cells (bottom right).
    Figure Legend Snippet: Differential effects of Akt inhibition at low and high concentrations of isoproterenol. (A) Floxed Akt2 fibroblasts stably expressing PPARγ were infected with either Adeno-GFP or Adeno-Cre and then differentiated into adipocytes. These cells were serum starved and treated with 1 nM insulin and analyzed by immunoblotting using Licor Odyssey for the excision of Akt2 and the loss of phospho-Akt Ser473 signal (left panel; representative of two experiments). The quantification of immunoblot analysis of phospho-Akt Ser473 of Akt2 lox/lox adipocytes infected with Ad-GFP or Ad-Cre is shown (middle panel). A glycerol release assay (right panel) was performed with increasing doses of isoproterenol in the presence and absence of 1 nM insulin for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. (B) RNA inteference-mediated reduction in Akt2 does not affect insulin inhibition of glycerol release. 3T3-L1 preadipocytes were infected with an shRNA lentiviral construct that targets Akt2 and sorted for the low and high expression of GFP. Adipocytes were treated with the indicated concentrations of insulin and subjected to the immunoblot analysis of Akt2 and phospho-Akt Thr308, confirming the efficiency of knockdown (left). Highly expressing cells were differentiated into adipocytes, and a glycerol release assay was performed using 2 nM isoproterenol with increasing doses of insulin (upper right). Data are expressed as means ± SD from two experiments performed in duplicate. A glucose uptake assay also was performed on differentiated control, highly, and lowly expressing cells (bottom right).

    Techniques Used: Inhibition, Stable Transfection, Expressing, Infection, Glucose Assay, shRNA, Construct

    6) Product Images from "Pharmacological Manipulation of the Akt Signaling Pathway Regulates Myxoma Virus Replication and Tropism in Human Cancer Cells ▿"

    Article Title: Pharmacological Manipulation of the Akt Signaling Pathway Regulates Myxoma Virus Replication and Tropism in Human Cancer Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02020-09

    Phospho status of Akt residue Thr308 mediates M-T5 binding. (A) Schematic representation of the Akt constructs used during this study. Amino acid substitutions are indicated. All Akt constructs contain an amino-terminal HA tag. (B) The plasmid MT5-GST
    Figure Legend Snippet: Phospho status of Akt residue Thr308 mediates M-T5 binding. (A) Schematic representation of the Akt constructs used during this study. Amino acid substitutions are indicated. All Akt constructs contain an amino-terminal HA tag. (B) The plasmid MT5-GST

    Techniques Used: Binding Assay, Construct, Plasmid Preparation

    Binding of M-T5 to cellular Akt induces phosphorylation of Akt and is a key restriction determinant for MYXV permissiveness in type II human cancer cells. Phosphorylation of Akt at residue Thr308 by PDK1 induces a conformational change to Akt, which allows
    Figure Legend Snippet: Binding of M-T5 to cellular Akt induces phosphorylation of Akt and is a key restriction determinant for MYXV permissiveness in type II human cancer cells. Phosphorylation of Akt at residue Thr308 by PDK1 induces a conformational change to Akt, which allows

    Techniques Used: Binding Assay

    7) Product Images from "Immobilization rapidly induces muscle insulin resistance together with the activation of MAPKs ( JNK and p38) and impairment of AS160 phosphorylation. Immobilization rapidly induces muscle insulin resistance together with the activation of MAPKs ( JNK and p38) and impairment of AS160 phosphorylation"

    Article Title: Immobilization rapidly induces muscle insulin resistance together with the activation of MAPKs ( JNK and p38) and impairment of AS160 phosphorylation. Immobilization rapidly induces muscle insulin resistance together with the activation of MAPKs ( JNK and p38) and impairment of AS160 phosphorylation

    Journal: Physiological Reports

    doi: 10.14814/phy2.12876

    Phosphorylations of Akt, AS 160, and TBC 1D1 in contralateral non‐immobilized and immobilized limbs at the end of 6‐h hindlimb immobilization. Muscles were dissected out at the end of 6‐h unilateral hindlimb immobilization. All muscles were incubated in glucose‐free medium in the absence or presence (50 μ U/ mL ) of insulin for 20 min and then frozen. Muscle lysates were separated with SDS ‐ PAGE and blots were analyzed for phosphorylated Akt Ser473 (A), phosphorylated Akt Thr308 (B), phosphorylated AS 160 Thr647 (C), phosphorylated AS 160 Ser588 (D), phosphorylated TBC 1D1 Thr590 (E), and phosphorylated TBC 1D1 Ser237 (F). Blots were then stripped and analyzed for total abundance of each protein. (A–C, F) Values are means ± SE ( n = 7–9). (D–E) Values are means ± SE ( n = 6–7). * P
    Figure Legend Snippet: Phosphorylations of Akt, AS 160, and TBC 1D1 in contralateral non‐immobilized and immobilized limbs at the end of 6‐h hindlimb immobilization. Muscles were dissected out at the end of 6‐h unilateral hindlimb immobilization. All muscles were incubated in glucose‐free medium in the absence or presence (50 μ U/ mL ) of insulin for 20 min and then frozen. Muscle lysates were separated with SDS ‐ PAGE and blots were analyzed for phosphorylated Akt Ser473 (A), phosphorylated Akt Thr308 (B), phosphorylated AS 160 Thr647 (C), phosphorylated AS 160 Ser588 (D), phosphorylated TBC 1D1 Thr590 (E), and phosphorylated TBC 1D1 Ser237 (F). Blots were then stripped and analyzed for total abundance of each protein. (A–C, F) Values are means ± SE ( n = 7–9). (D–E) Values are means ± SE ( n = 6–7). * P

    Techniques Used: Incubation, SDS Page

    8) Product Images from "A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines"

    Article Title: A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-016-1129-6

    FVE-induced autophagy. a Accumulation of GFP-LC3 punctate vesicles in MDA-MB-231 cells. (b1 and c1) FVE reduced Akt phosphorylation at Ser473 and Thr308 in MCF-7 and MDA-MB-231 cells. (b2 and c2) FVE decreased PI3K, 4-EB-P1 and p70S6K phosphorylation in MCF-7 and MDA-MB-231 cells. (b3 and c3) FVE promoted accumulation of phospho-Beclin 1 and LC3B II in MCF-7 and MDA-MB-231 cells
    Figure Legend Snippet: FVE-induced autophagy. a Accumulation of GFP-LC3 punctate vesicles in MDA-MB-231 cells. (b1 and c1) FVE reduced Akt phosphorylation at Ser473 and Thr308 in MCF-7 and MDA-MB-231 cells. (b2 and c2) FVE decreased PI3K, 4-EB-P1 and p70S6K phosphorylation in MCF-7 and MDA-MB-231 cells. (b3 and c3) FVE promoted accumulation of phospho-Beclin 1 and LC3B II in MCF-7 and MDA-MB-231 cells

    Techniques Used: Multiple Displacement Amplification

    9) Product Images from "Hepatocyte Nuclear Factor 1A (HNF1A) as a Possible Tumor Suppressor in Pancreatic Cancer"

    Article Title: Hepatocyte Nuclear Factor 1A (HNF1A) as a Possible Tumor Suppressor in Pancreatic Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121082

    Effect of HNF1A knockdown on AKT/mTOR signaling pathway was assessed by Western Blot. AsPC-1 and BxPC-3 cells were transfected with control siRNA and two different HNF1A siRNAs as indicated. 96h after transfection, cell lysates were immunoblotted with anti- HNF1A antibody, anti-phospho-AKT Ser473 antibody, anti-phospho-AKT Thr308 antibody, and anti-phospho-mTOR Ser2448 antibody. Membranes were stripped and re-probed with anti-AKT antibody, anti-mTOR antibody and anti-β-actin antibody. The fold differences in protein expression levels of cells transfected with HNF1A siRNA and control siRNA was presented as mean ± SD from three independent experiments.
    Figure Legend Snippet: Effect of HNF1A knockdown on AKT/mTOR signaling pathway was assessed by Western Blot. AsPC-1 and BxPC-3 cells were transfected with control siRNA and two different HNF1A siRNAs as indicated. 96h after transfection, cell lysates were immunoblotted with anti- HNF1A antibody, anti-phospho-AKT Ser473 antibody, anti-phospho-AKT Thr308 antibody, and anti-phospho-mTOR Ser2448 antibody. Membranes were stripped and re-probed with anti-AKT antibody, anti-mTOR antibody and anti-β-actin antibody. The fold differences in protein expression levels of cells transfected with HNF1A siRNA and control siRNA was presented as mean ± SD from three independent experiments.

    Techniques Used: Western Blot, Transfection, Expressing

    10) Product Images from "Full-Length Adiponectin Attenuates Insulin Signaling and Inhibits Insulin-Stimulated Amino Acid Transport in Human Primary Trophoblast Cells"

    Article Title: Full-Length Adiponectin Attenuates Insulin Signaling and Inhibits Insulin-Stimulated Amino Acid Transport in Human Primary Trophoblast Cells

    Journal: Diabetes

    doi: 10.2337/db09-0824

    Representative Western blots ( A ) and summary data ( B ) of phospho-AKT Ser473 and phospho-AKT Thr308 protein expression after incubation of cultured trophoblast cells with control media, insulin (1 nmol/l), gAd (5 μg/ml), or fAd (5 μg/ml) for 24 h. Subsets of cells were pretreated with insulin (1 nmol/l) for 4 h and then exposed to 5 μg/ml gAd (IgAd) or fAd (IfAd) for an additional 20 h. n = 5 placentas for each treatment. Insulin significantly increased expression of both phospho-AKT Ser473 ( P
    Figure Legend Snippet: Representative Western blots ( A ) and summary data ( B ) of phospho-AKT Ser473 and phospho-AKT Thr308 protein expression after incubation of cultured trophoblast cells with control media, insulin (1 nmol/l), gAd (5 μg/ml), or fAd (5 μg/ml) for 24 h. Subsets of cells were pretreated with insulin (1 nmol/l) for 4 h and then exposed to 5 μg/ml gAd (IgAd) or fAd (IfAd) for an additional 20 h. n = 5 placentas for each treatment. Insulin significantly increased expression of both phospho-AKT Ser473 ( P

    Techniques Used: Western Blot, Expressing, Incubation, Cell Culture

    11) Product Images from "Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes"

    Article Title: Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes

    Journal: Archives of Physiology and Biochemistry

    doi: 10.3109/13813455.2013.774022

    Effect of prednisolone on insulin action in 3T3-L1 adipocyte cells. Effects on protein expression of the insulin receptor (IR) β-subunit, Akt, AS160, and GLUT4, as well as the phosphorylation of insulin receptor substrate 1 (IRS1-Tyr1222), Akt-Thr308, Akt-Ser473, and AS160-Thr642 were determined by Western blotting in cells exposed to 1 μM prednisolone for 48 h prior to stimulation with insulin (10 min, 100 nM). Data are presented as mean ± standard error of the mean of > 4 independent experiments (A–D), and representative Western blots (F). E. Effect of prednisolone on 2-deoxyglucose uptake in in 3T3-L1 adipocytes after 6, 24 and 48 h of incubation with prednisolone. Data are mean ± standard error of the mean of > 4 independent experiments. In all bar graphs, open columns represent the basal condition, and filled bars depict insulin-stimulated cells. The effects of prednisolone on insulin action were analysed using a two-way ANOVA followed by Bonferonni analysis for multiple comparisons. ***Indicates a p
    Figure Legend Snippet: Effect of prednisolone on insulin action in 3T3-L1 adipocyte cells. Effects on protein expression of the insulin receptor (IR) β-subunit, Akt, AS160, and GLUT4, as well as the phosphorylation of insulin receptor substrate 1 (IRS1-Tyr1222), Akt-Thr308, Akt-Ser473, and AS160-Thr642 were determined by Western blotting in cells exposed to 1 μM prednisolone for 48 h prior to stimulation with insulin (10 min, 100 nM). Data are presented as mean ± standard error of the mean of > 4 independent experiments (A–D), and representative Western blots (F). E. Effect of prednisolone on 2-deoxyglucose uptake in in 3T3-L1 adipocytes after 6, 24 and 48 h of incubation with prednisolone. Data are mean ± standard error of the mean of > 4 independent experiments. In all bar graphs, open columns represent the basal condition, and filled bars depict insulin-stimulated cells. The effects of prednisolone on insulin action were analysed using a two-way ANOVA followed by Bonferonni analysis for multiple comparisons. ***Indicates a p

    Techniques Used: Expressing, Western Blot, Incubation

    12) Product Images from "Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells"

    Article Title: Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082625

    Effect of androgen treatment on protein abundance of AR, PSA, and cell cycle regulators in LNCaP sublines, DU-145, and PC-3 cells. Protein expression of total Akt, Akt1, Akt2, phospho-Akt Ser473, phospho-Akt Thr308, p42/44 MAPK, phospho-p42/44 MAPK Thr202/Tyr204, EGFR, and different phosphorylation site of tyrosine (Tyr1173, Tyr1148, Tyr1086, Tyr1069, Tyr1045, and Tyr845) in LNCaP 104-S, 104-R1, and 104-R2 cells treated with 0, 0.1, or 10 nM R1881 for 96 hours were assayed by Western blotting. Same GAPDH, α-tubulin, and β-actin as shown in Figure 3 were used as loading control. These proteins were also examined in PC-3 and DU-145 cells in the absence of androgen. Experiments were repeated three times. Numbers represent quantification of bands of individual protein quantified by ImageJ.
    Figure Legend Snippet: Effect of androgen treatment on protein abundance of AR, PSA, and cell cycle regulators in LNCaP sublines, DU-145, and PC-3 cells. Protein expression of total Akt, Akt1, Akt2, phospho-Akt Ser473, phospho-Akt Thr308, p42/44 MAPK, phospho-p42/44 MAPK Thr202/Tyr204, EGFR, and different phosphorylation site of tyrosine (Tyr1173, Tyr1148, Tyr1086, Tyr1069, Tyr1045, and Tyr845) in LNCaP 104-S, 104-R1, and 104-R2 cells treated with 0, 0.1, or 10 nM R1881 for 96 hours were assayed by Western blotting. Same GAPDH, α-tubulin, and β-actin as shown in Figure 3 were used as loading control. These proteins were also examined in PC-3 and DU-145 cells in the absence of androgen. Experiments were repeated three times. Numbers represent quantification of bands of individual protein quantified by ImageJ.

    Techniques Used: Expressing, Western Blot

    13) Product Images from "Akt/PKB Controls the Activity-Dependent Bulk Endocytosis of Synaptic Vesicles"

    Article Title: Akt/PKB Controls the Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Journal: Traffic (Copenhagen, Denmark)

    doi: 10.1111/j.1600-0854.2012.01365.x

    Akt is phosphorylated in an activity-dependent manner Cultures were subjected to action potential trains of increasing frequency (10, 20, 40 or 80 Hz) for 10 seconds. The extent of phosphorylation of either Akt Ser473 [Pser, (A)] or Thr308 [PThr (C)] was assessed by western blotting. Synaptophysin (Syp) blots were performed as loading controls. Representative blots are displayed for all experiments. The extent of phosphorylation of either Akt Ser473 (B) or Thr308 (D) is displayed. Data were corrected against protein levels (Syp) and then normalized to basal ± SEM ( n = 7 for PSer Akt and n = 5 for PThr Akt). One-way anova : *p
    Figure Legend Snippet: Akt is phosphorylated in an activity-dependent manner Cultures were subjected to action potential trains of increasing frequency (10, 20, 40 or 80 Hz) for 10 seconds. The extent of phosphorylation of either Akt Ser473 [Pser, (A)] or Thr308 [PThr (C)] was assessed by western blotting. Synaptophysin (Syp) blots were performed as loading controls. Representative blots are displayed for all experiments. The extent of phosphorylation of either Akt Ser473 (B) or Thr308 (D) is displayed. Data were corrected against protein levels (Syp) and then normalized to basal ± SEM ( n = 7 for PSer Akt and n = 5 for PThr Akt). One-way anova : *p

    Techniques Used: Activity Assay, Western Blot

    14) Product Images from "The Dichotomy of Endoplasmic Reticulum Stress Response in Liver Ischemia Reperfusion Injury"

    Article Title: The Dichotomy of Endoplasmic Reticulum Stress Response in Liver Ischemia Reperfusion Injury

    Journal: Transplantation

    doi: 10.1097/TP.0000000000001032

    Autophagy signaling pathways and machinery proteins in liver IRI. Liver tissues were harvested after either 30m or 90m ischemia and 0h or 1h of reperfusion. (A) Representative Western blots of phosphorylated Akt (at Thr308 and Ser473), phosphorylated
    Figure Legend Snippet: Autophagy signaling pathways and machinery proteins in liver IRI. Liver tissues were harvested after either 30m or 90m ischemia and 0h or 1h of reperfusion. (A) Representative Western blots of phosphorylated Akt (at Thr308 and Ser473), phosphorylated

    Techniques Used: Western Blot

    15) Product Images from "Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability"

    Article Title: Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147011

    Effect of Cd on Akt phosphorylaion at different sites. HepG2 cells were treated with 50 μM of PI3K ((LY294002), p38 (SB202190) or JNK (SP600125) inhibitor 1 h prior to the addition of 5 μM Cd. Cells were cultured for additional 4 h then harvested and prepared for Western blotting. Antibodies raised specifically against phosphorylation at Thr308 [p-Akt(T308)], Thr450 [p-Akt(T450)] or Ser473 [p-Akt(S473)] of Akt were used for the analysis. Actin was used as a loading control for immunoblotting.
    Figure Legend Snippet: Effect of Cd on Akt phosphorylaion at different sites. HepG2 cells were treated with 50 μM of PI3K ((LY294002), p38 (SB202190) or JNK (SP600125) inhibitor 1 h prior to the addition of 5 μM Cd. Cells were cultured for additional 4 h then harvested and prepared for Western blotting. Antibodies raised specifically against phosphorylation at Thr308 [p-Akt(T308)], Thr450 [p-Akt(T450)] or Ser473 [p-Akt(S473)] of Akt were used for the analysis. Actin was used as a loading control for immunoblotting.

    Techniques Used: Cell Culture, Western Blot

    16) Product Images from "Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle"

    Article Title: Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    doi: 10.1152/ajpregu.00659.2010

    Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), Akt on Thr308 ( B ), and Ser473 ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined
    Figure Legend Snippet: Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), Akt on Thr308 ( B ), and Ser473 ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined

    Techniques Used:

    17) Product Images from "Systemic insulin sensitivity is regulated by GPS2 inhibition of AKT ubiquitination and activation in adipose tissue"

    Article Title: Systemic insulin sensitivity is regulated by GPS2 inhibition of AKT ubiquitination and activation in adipose tissue

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2016.10.007

    AKT ubiquitination and activation upon insulin stimulation is regulated by the opposing actions of the ubiquitin conjugating enzyme Ubc13 and its inhibitor GPS2. (A) Detection of K63 ubiquitination by IP/WB of AKT in 3T3-L1 preadipocytes serum starved overnight prior to stimulation with 100 nM insulin. Phosphorylation of AKT on Thr308 is measured in parallel on whole cell extracts from the same cells and normalized to total AKT levels. (B) Inducible AKT1 and AKT2 deletion upon 4-hydroxy tamoxifen treatment (4OH-Tam) is rescued by transient overexpression of AKT2 WT or ubiquitination mutants. Protein expression levels and insulin mediated activation of AKT2 and downstream effectors PRAS40 and ribosomal protein S6 are assayed by WB in whole cell extracts. (C) WB analysis of phospho-AKT/total AKT on whole cell extracts from siCTRL versus siUBC13 transiently transfected 3T3-L1 preadipocytes. Cells were stimulated with insulin (5′) after overnight serum starvation. (D) WB analysis of phospho-AKT/total AKT and phospho-GSK3B/total GSK3B in cytosolic cell extracts from proliferating 3T3-L1 preadipocytes transiently transfected with siCTRL (WT) or siGPS2 (GPS2-KD). (E) WB analysis of phospho-AKT/total AKT in whole cell extracts from 293T cells. (F) WB analysis of phospho-AKT/total AKT in whole cell extracts from WT and GPS2-KD 3T3-L1 preadipocytes starved overnight prior to insulin stimulation (3′). All western blots are representative images of at least three independent experiments.
    Figure Legend Snippet: AKT ubiquitination and activation upon insulin stimulation is regulated by the opposing actions of the ubiquitin conjugating enzyme Ubc13 and its inhibitor GPS2. (A) Detection of K63 ubiquitination by IP/WB of AKT in 3T3-L1 preadipocytes serum starved overnight prior to stimulation with 100 nM insulin. Phosphorylation of AKT on Thr308 is measured in parallel on whole cell extracts from the same cells and normalized to total AKT levels. (B) Inducible AKT1 and AKT2 deletion upon 4-hydroxy tamoxifen treatment (4OH-Tam) is rescued by transient overexpression of AKT2 WT or ubiquitination mutants. Protein expression levels and insulin mediated activation of AKT2 and downstream effectors PRAS40 and ribosomal protein S6 are assayed by WB in whole cell extracts. (C) WB analysis of phospho-AKT/total AKT on whole cell extracts from siCTRL versus siUBC13 transiently transfected 3T3-L1 preadipocytes. Cells were stimulated with insulin (5′) after overnight serum starvation. (D) WB analysis of phospho-AKT/total AKT and phospho-GSK3B/total GSK3B in cytosolic cell extracts from proliferating 3T3-L1 preadipocytes transiently transfected with siCTRL (WT) or siGPS2 (GPS2-KD). (E) WB analysis of phospho-AKT/total AKT in whole cell extracts from 293T cells. (F) WB analysis of phospho-AKT/total AKT in whole cell extracts from WT and GPS2-KD 3T3-L1 preadipocytes starved overnight prior to insulin stimulation (3′). All western blots are representative images of at least three independent experiments.

    Techniques Used: Activation Assay, Western Blot, Over Expression, Expressing, Transfection

    18) Product Images from "Exercise training is an effective alternative to estrogen supplementation for improving glucose homeostasis in ovariectomized rats"

    Article Title: Exercise training is an effective alternative to estrogen supplementation for improving glucose homeostasis in ovariectomized rats

    Journal: Physiological Reports

    doi: 10.14814/phy2.12617

    Protein content of insulin signaling markers before (basal) and after 7.5 U/kg b.w injection of insulin in visceral adipose tissue (VAT) p-Akt Ser473 and Thr308 (A, B) and subcutaneous adipose tissue (SC) p-Akt Ser473 and Thr308 (C, D). Data are expressed as mean ± standard error, n = 10 per group. Groups which share a letter are not significantly different. Statistical significance is accepted at P
    Figure Legend Snippet: Protein content of insulin signaling markers before (basal) and after 7.5 U/kg b.w injection of insulin in visceral adipose tissue (VAT) p-Akt Ser473 and Thr308 (A, B) and subcutaneous adipose tissue (SC) p-Akt Ser473 and Thr308 (C, D). Data are expressed as mean ± standard error, n = 10 per group. Groups which share a letter are not significantly different. Statistical significance is accepted at P

    Techniques Used: Injection

    Protein content of insulin signaling markers before (basal) and after (+insulin) 7.5 U/kg b.w injection of insulin in liver (VAT) p-Akt Ser473 and Thr308 (A, B) and total Akt (C). Data are expressed as mean ± standard error, n = 10 per group. There were no statistical differences in content between groups on either Akt reside. Statistical significance is accepted at P
    Figure Legend Snippet: Protein content of insulin signaling markers before (basal) and after (+insulin) 7.5 U/kg b.w injection of insulin in liver (VAT) p-Akt Ser473 and Thr308 (A, B) and total Akt (C). Data are expressed as mean ± standard error, n = 10 per group. There were no statistical differences in content between groups on either Akt reside. Statistical significance is accepted at P

    Techniques Used: Injection

    Basal and maximally insulin-stimulated glucose uptake in soleus and EDL (A, D), protein content of insulin signaling markers before (basal) and after (+insulin) 7.5 U/kg b.w injection in soleus p-Akt Ser 473 and p-Akt Thr308 (B, C) and EDL p-Akt Ser473 and p-Akt Thr308 (E, F). Data are expressed as mean ± standard error, n = 10 per group. Groups which share a letter are not significantly different. In (A), bars with an asterisk denote a significant insulin effect relative to the basal control within each group. Statistical significance is accepted at P
    Figure Legend Snippet: Basal and maximally insulin-stimulated glucose uptake in soleus and EDL (A, D), protein content of insulin signaling markers before (basal) and after (+insulin) 7.5 U/kg b.w injection in soleus p-Akt Ser 473 and p-Akt Thr308 (B, C) and EDL p-Akt Ser473 and p-Akt Thr308 (E, F). Data are expressed as mean ± standard error, n = 10 per group. Groups which share a letter are not significantly different. In (A), bars with an asterisk denote a significant insulin effect relative to the basal control within each group. Statistical significance is accepted at P

    Techniques Used: Injection

    19) Product Images from "Leucine and HMB Differentially Modulate Proteasome System in Skeletal Muscle under Different Sarcopenic Conditions"

    Article Title: Leucine and HMB Differentially Modulate Proteasome System in Skeletal Muscle under Different Sarcopenic Conditions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076752

    Effect of HMB and leucine under PI3K/AKT protein levels during atrophic stimuli. Representative protein level of PI3K, AKT total, AKT phosphorilation residues at Thr308 and Ser473 and 4E-BP1, 3 days after dexamethasone administration or hind limb immobilization, under leucine (Dexa+Leu and Imob+Leu, respectively) or HMB (Dexa+HMB and Imob+HMB, respectively). Each pair of lanes represents a duplicate of each group (n=4 per group). Sarcomeric actin was used as loading control. The bars in B and C represent mean±S.D. a -p
    Figure Legend Snippet: Effect of HMB and leucine under PI3K/AKT protein levels during atrophic stimuli. Representative protein level of PI3K, AKT total, AKT phosphorilation residues at Thr308 and Ser473 and 4E-BP1, 3 days after dexamethasone administration or hind limb immobilization, under leucine (Dexa+Leu and Imob+Leu, respectively) or HMB (Dexa+HMB and Imob+HMB, respectively). Each pair of lanes represents a duplicate of each group (n=4 per group). Sarcomeric actin was used as loading control. The bars in B and C represent mean±S.D. a -p

    Techniques Used:

    20) Product Images from "Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse. Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse"

    Article Title: Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse. Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse

    Journal: Physiological Reports

    doi: 10.14814/phy2.13800

    Lactate administration increases intracellular signaling related to ERK 1/2 and Akt/ mTORC 1 pathways. Phosphorylation of ERK 1/2 Thr202/Tyr204 (A), IGF ‐1 R T yr1135/1136 (B), Akt Thr308 (C), Akt Ser473 (D), p70S6 K T hr389 (E), and rpS6 Ser235/236 (F) in quadriceps (Quad), extensor digitorum longus ( EDL ), and soleus (Sol) skeletal muscles after vehicle ( VEH , n = 5) or lactate ( LAC , n = 7) administration. Bars represent the median in the scatterplot. Representative immunoblots are shown in G. VEH , vehicle; LAC , lactate. Values in graphs are arbitrary units (A.U.). Statistical significance compared with vehicle is indicated by * P
    Figure Legend Snippet: Lactate administration increases intracellular signaling related to ERK 1/2 and Akt/ mTORC 1 pathways. Phosphorylation of ERK 1/2 Thr202/Tyr204 (A), IGF ‐1 R T yr1135/1136 (B), Akt Thr308 (C), Akt Ser473 (D), p70S6 K T hr389 (E), and rpS6 Ser235/236 (F) in quadriceps (Quad), extensor digitorum longus ( EDL ), and soleus (Sol) skeletal muscles after vehicle ( VEH , n = 5) or lactate ( LAC , n = 7) administration. Bars represent the median in the scatterplot. Representative immunoblots are shown in G. VEH , vehicle; LAC , lactate. Values in graphs are arbitrary units (A.U.). Statistical significance compared with vehicle is indicated by * P

    Techniques Used: Western Blot

    21) Product Images from "Discoidin Domain Receptor 2 Signaling Regulates Fibroblast Apoptosis through PDK1/Akt"

    Article Title: Discoidin Domain Receptor 2 Signaling Regulates Fibroblast Apoptosis through PDK1/Akt

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2017-0419OC

    DDR2 regulates fibroblast Akt pathway. ( A ) IB for phospho-extracellular signal-regulated kinase (p-ERK), ERK, phospho-Src (p-Src), Src, and XIAP (X-linked inhibitor of apoptosis protein) in primary lung fibroblasts isolated from DDR2-null (null) and littermate control (WT) mice. ( B ) IB for phospho-Ser473-Akt (pSer473-Akt), phospho-Thr308-Akt (pThr308-Akt), and total Akt in primary lung fibroblasts isolated from null and WT mice.
    Figure Legend Snippet: DDR2 regulates fibroblast Akt pathway. ( A ) IB for phospho-extracellular signal-regulated kinase (p-ERK), ERK, phospho-Src (p-Src), Src, and XIAP (X-linked inhibitor of apoptosis protein) in primary lung fibroblasts isolated from DDR2-null (null) and littermate control (WT) mice. ( B ) IB for phospho-Ser473-Akt (pSer473-Akt), phospho-Thr308-Akt (pThr308-Akt), and total Akt in primary lung fibroblasts isolated from null and WT mice.

    Techniques Used: Isolation, Mouse Assay

    22) Product Images from "HLA Class II Triggered Signaling Cascades Cause Endothelial Cell Proliferation and Migration: Relevance to Antibody-Mediated Transplant Rejection 1"

    Article Title: HLA Class II Triggered Signaling Cascades Cause Endothelial Cell Proliferation and Migration: Relevance to Antibody-Mediated Transplant Rejection 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1701259

    ERK1/2 regulates HLA class II antibody-induced activation of intracellular signal transduction networks, cell proliferation, and migration EC were A, C, E, G, I infected with Ad-LacZ or Ad-CIITA or B, D, F, H, J pretreated with TNFα/IFNγ for 48 h. A, B Starved cells were pretreated with 5 μM UO126, or E, F, G, H, I, J with 1.0 μM UO126, or A, B, with 5.0 μM PD0325901, or E, F with 1.0 μM PD0325901, or G, H, I, J with 1.0 μM UO124 for 60 min, or C, D, Starved EC were transfected with 100 nM of MEK, or control siRNA for 48 hours. A, B, C, D Pretreated ECwere stimulated with0.1 μg/ml of HLA class II antibody for 15 min. Treatment of EC with mIgG serves as a negative control. Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting with anti-phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6K Thr421/424, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin antibodies to confirm equal loading of proteins. E, F Cells were stimulated with 1.0 μg/ml HLA class II antibody for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. G, H Cells were pretreated with 10 μg/ml of mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1.0 μg/ml of anti-HLA class II antibody for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of HLA class II antibody-stimulated EC divided by the cell number between two initiated front edges of negative control EC. I, J Cell migration was measured in a transwell insert system. EC infected with Ad-LacZ or Ad-CIITA or pretreated with TNFα/IFNγwere seeded to the upper chamber of insert, pretreated with UO126 or UO124 for 60 min, and stimulated with 1.0 μg/ml HLA class II antibody for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with 10 × objective lens, and migrated cells were counted. The bar graphs show the mean ± SEM fold change of C, D. proliferation index, E, F, G, H migrated cells. *p
    Figure Legend Snippet: ERK1/2 regulates HLA class II antibody-induced activation of intracellular signal transduction networks, cell proliferation, and migration EC were A, C, E, G, I infected with Ad-LacZ or Ad-CIITA or B, D, F, H, J pretreated with TNFα/IFNγ for 48 h. A, B Starved cells were pretreated with 5 μM UO126, or E, F, G, H, I, J with 1.0 μM UO126, or A, B, with 5.0 μM PD0325901, or E, F with 1.0 μM PD0325901, or G, H, I, J with 1.0 μM UO124 for 60 min, or C, D, Starved EC were transfected with 100 nM of MEK, or control siRNA for 48 hours. A, B, C, D Pretreated ECwere stimulated with0.1 μg/ml of HLA class II antibody for 15 min. Treatment of EC with mIgG serves as a negative control. Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting with anti-phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6K Thr421/424, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin antibodies to confirm equal loading of proteins. E, F Cells were stimulated with 1.0 μg/ml HLA class II antibody for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry. DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percentage of cells positive for BrdU normalized to negative control. G, H Cells were pretreated with 10 μg/ml of mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1.0 μg/ml of anti-HLA class II antibody for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of HLA class II antibody-stimulated EC divided by the cell number between two initiated front edges of negative control EC. I, J Cell migration was measured in a transwell insert system. EC infected with Ad-LacZ or Ad-CIITA or pretreated with TNFα/IFNγwere seeded to the upper chamber of insert, pretreated with UO126 or UO124 for 60 min, and stimulated with 1.0 μg/ml HLA class II antibody for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with 10 × objective lens, and migrated cells were counted. The bar graphs show the mean ± SEM fold change of C, D. proliferation index, E, F, G, H migrated cells. *p

    Techniques Used: Activation Assay, Transduction, Migration, Infection, Transfection, Negative Control, SDS Page, Flow Cytometry, Cytometry, DNA Synthesis, Transferring, Incubation, Staining

    Src regulates HLA class II antibody-stimulated activation of intracellular signal networks, cell proliferation, and migration EC were A, C, E, G, I, K infected with Ad-LacZ or Ad-CIITA or B, D, F, H, J, L pretreated with TNFα/IFNγ for 48 h. A, B, C, D, G, H, I, J, K, L Starved cells were pretreated with 12.5 μM PP2, or A, B, G, H with 1.0 μM dasatinib, or I, J, K, L with PP2 inactive analog PP3 for 30 min. E, F, Starved EC were transfected with 100 nM of c-Src, or control siRNA for 48 hours. A, B, E, F EC were stimulated with0.1 μg/ml HLA class II antibody or mIgG control for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting withanti-phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin antibodies to confirm equal loading of proteins. C, D EC were detached, incubated with 1.0 μg/ml HLA class II antibody for 30 min at 4°C, after wash 3 times with PFA the cells were incubated with FITC-conjugated goat anti-mouse F(ab’)2 fragment for 30 min at 4°C. The fluorescence intensity was measured by LSRFortessa flow cytometry and calculated using FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one way ANOVA with Fisher's LSD. G, H Starved cells were stimulated with 1.0 μg/ml HLA class II antibody for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percent of cells positive for BrdU normalized to negative control. I, J Cells were pretreated with 10 μg/ml of mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1.0 μg/ml of anti-HLA class II antibody for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II-stimulated EC divided by the cell number between two initiated front edges of negative control EC. K, L Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNFα/IFNγwere seeded to the upper chamber of insert and stimulated with 1.0 μg/ml HLA class II antibody for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with 10 × objective lens, and migrated cells were counted. G, H The bar graphs show the mean ± SEM fold change of proliferation index. I, J, K, L The bar graph shows the mean ± SEM number of migrated cells. **p
    Figure Legend Snippet: Src regulates HLA class II antibody-stimulated activation of intracellular signal networks, cell proliferation, and migration EC were A, C, E, G, I, K infected with Ad-LacZ or Ad-CIITA or B, D, F, H, J, L pretreated with TNFα/IFNγ for 48 h. A, B, C, D, G, H, I, J, K, L Starved cells were pretreated with 12.5 μM PP2, or A, B, G, H with 1.0 μM dasatinib, or I, J, K, L with PP2 inactive analog PP3 for 30 min. E, F, Starved EC were transfected with 100 nM of c-Src, or control siRNA for 48 hours. A, B, E, F EC were stimulated with0.1 μg/ml HLA class II antibody or mIgG control for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting withanti-phospho-Src Tyr418, FAK Tyr576, FAK Tyr577, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, ERK, or β-actin antibodies to confirm equal loading of proteins. C, D EC were detached, incubated with 1.0 μg/ml HLA class II antibody for 30 min at 4°C, after wash 3 times with PFA the cells were incubated with FITC-conjugated goat anti-mouse F(ab’)2 fragment for 30 min at 4°C. The fluorescence intensity was measured by LSRFortessa flow cytometry and calculated using FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one way ANOVA with Fisher's LSD. G, H Starved cells were stimulated with 1.0 μg/ml HLA class II antibody for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percent of cells positive for BrdU normalized to negative control. I, J Cells were pretreated with 10 μg/ml of mitomycin C for 2 h to inhibit cell proliferation before being assayed for their ability to migrate. A scratch wound was created with a sterile 200-μl pipette tip. Wounded cells were stimulated with 1.0 μg/ml of anti-HLA class II antibody for 16 h. The cell number between two initiated front edges was counted; migration rate was analyzed by calculating the cell number between two initiated front edges of class II-stimulated EC divided by the cell number between two initiated front edges of negative control EC. K, L Cell migration was measured in a transwell insert system. EC infected with Ad-CIITA or pretreated with TNFα/IFNγwere seeded to the upper chamber of insert and stimulated with 1.0 μg/ml HLA class II antibody for 16 h at 37°C. After incubation, the cells on the upper surface of the insert membrane were removed with a cotton swab, the migrated cells on the bottom of the insert membrane were fixed with methanol and stained with crystal violet, three middle fields per insert were photographed with 10 × objective lens, and migrated cells were counted. G, H The bar graphs show the mean ± SEM fold change of proliferation index. I, J, K, L The bar graph shows the mean ± SEM number of migrated cells. **p

    Techniques Used: Activation Assay, Migration, Infection, Transfection, Negative Control, SDS Page, Incubation, Fluorescence, Flow Cytometry, Cytometry, Expressing, DNA Synthesis, Transferring, Staining

    mTOR regulates HLA class II antibody-stimulated activation of intracellular signal networks, cell proliferation and migration EC wereinfected with Ad-LacZ A, C, I or Ad-CIITA or B, D, E, F, J pretreated with TNFα/IFNγ for 48 h. A, B, E, F StarvedEC were pretreated with 30 nM of rapamycin for 2 h or 24 h, or E, F with 10 nM of everolimus for 2 h or 24 h;or I, J with 30 nM of rapamycin or 10 nM of everolimus for 24 h; or E, F, I, J with 1.0 μM of PP242 for 2 h. A, B, E, F Cells were stimulated with 0.1 μg/ml HLA II antibody or control mIgG for 15 min. Proteins in the precleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting with A, B anti-phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, A, B, E, F Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK1/2 Thr202/Tyr204 antibodies. The membrane was re-probed with anti-vinculin, S6K, S6RP, Akt, ERK or A, B Src, FAK, PI3K, mTOR, or β-actin total antibodies to confirm equal loading of proteins. C, D, G, H The bar graphs show phosphorylated protein bands shown in A, B, E, F were quantified by densitometry scan analysis, and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. *p
    Figure Legend Snippet: mTOR regulates HLA class II antibody-stimulated activation of intracellular signal networks, cell proliferation and migration EC wereinfected with Ad-LacZ A, C, I or Ad-CIITA or B, D, E, F, J pretreated with TNFα/IFNγ for 48 h. A, B, E, F StarvedEC were pretreated with 30 nM of rapamycin for 2 h or 24 h, or E, F with 10 nM of everolimus for 2 h or 24 h;or I, J with 30 nM of rapamycin or 10 nM of everolimus for 24 h; or E, F, I, J with 1.0 μM of PP242 for 2 h. A, B, E, F Cells were stimulated with 0.1 μg/ml HLA II antibody or control mIgG for 15 min. Proteins in the precleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting with A, B anti-phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, A, B, E, F Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK1/2 Thr202/Tyr204 antibodies. The membrane was re-probed with anti-vinculin, S6K, S6RP, Akt, ERK or A, B Src, FAK, PI3K, mTOR, or β-actin total antibodies to confirm equal loading of proteins. C, D, G, H The bar graphs show phosphorylated protein bands shown in A, B, E, F were quantified by densitometry scan analysis, and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. *p

    Techniques Used: Activation Assay, Migration, SDS Page

    Effects of mTOR, Raptor, and Rictor siRNA knockdown on HLA II antibody-stimulated EC proliferation and activation of signal transduction networks A, B, C, D, E EC were transfected with 100 nM mTOR, or A, B, C, D, E, F, G Raptor, Rictor, or control siRNA for 48 h. A, F, G Protein in pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting with anti-mTOR, Raptor, Rictor, S6K, Akt, ERK, Vinculin, β-tubulin, and β-actin. B, D, F EC wereinfected with Ad-LacZ or Ad-CIITA or C, E, G pretreated with TNFα/IFNγ for 48 hours. B, C EC were detached with 0.125% trypsin/0.05% EDTA, washed with PFA, and incubated with 1.0 μg/ml HLA class II antibody for 30 min at 4°C. EC were washed 3 times with PFA and incubated with FITC-conjugated goat anti-mouse F(ab’)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on anLSRFortessa flow cytometer and calculated using the FACSDiva program (Becton Dickinson, Mountain View, CA). The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one way ANOVA with Fisher's LSD. D, E Transfected EC were stimulated with 1.0 μg/ml HLA class II antibody or mIgG for 48 h. EC were incorporated with BrdU for 2 h and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percent of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. F, G Transfected ECwere stimulated with0.1 μg/ml HLA class II antibody for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting withantibodies to Raptor, Rictor for knockdown efficiency, anti-phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Src, FAK, PI3K, Akt, S6K, S6RP, ERK, or Vinculin antibodies to confirm equal loading of proteins. H, I Phosphorylated protein bands shown in F and G were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. ***p
    Figure Legend Snippet: Effects of mTOR, Raptor, and Rictor siRNA knockdown on HLA II antibody-stimulated EC proliferation and activation of signal transduction networks A, B, C, D, E EC were transfected with 100 nM mTOR, or A, B, C, D, E, F, G Raptor, Rictor, or control siRNA for 48 h. A, F, G Protein in pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting with anti-mTOR, Raptor, Rictor, S6K, Akt, ERK, Vinculin, β-tubulin, and β-actin. B, D, F EC wereinfected with Ad-LacZ or Ad-CIITA or C, E, G pretreated with TNFα/IFNγ for 48 hours. B, C EC were detached with 0.125% trypsin/0.05% EDTA, washed with PFA, and incubated with 1.0 μg/ml HLA class II antibody for 30 min at 4°C. EC were washed 3 times with PFA and incubated with FITC-conjugated goat anti-mouse F(ab’)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on anLSRFortessa flow cytometer and calculated using the FACSDiva program (Becton Dickinson, Mountain View, CA). The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one way ANOVA with Fisher's LSD. D, E Transfected EC were stimulated with 1.0 μg/ml HLA class II antibody or mIgG for 48 h. EC were incorporated with BrdU for 2 h and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index (PI) is presented as fold increase in the percent of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. F, G Transfected ECwere stimulated with0.1 μg/ml HLA class II antibody for 15 min. Treatment of EC with mIgG serves as negative control. Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting withantibodies to Raptor, Rictor for knockdown efficiency, anti-phospho-Src Tyr418, FAK Tyr576, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, S6K Thr389, S6RP Ser240/244, c-Raf-1 Ser259, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Src, FAK, PI3K, Akt, S6K, S6RP, ERK, or Vinculin antibodies to confirm equal loading of proteins. H, I Phosphorylated protein bands shown in F and G were quantified by densitometry scan analysis and results are expressed as the mean ± SEM percentage of maximal increase in phosphorylation above control values. ***p

    Techniques Used: Activation Assay, Transduction, Transfection, SDS Page, Incubation, Fluorescence, Flow Cytometry, Cytometry, Expressing, DNA Synthesis, Negative Control

    Effect of FAK siRNA transfection on HLA II antibody-stimulated activation of signal transduction networks and cell proliferation A, D, F EC were infected with Ad-LacZ or Ad-CIITA or B, E, G pretreated with TNFα/IFNγ for 48 hours. A, B, C, D, E, F, G Starved EC were transfected with 100 nM of FAK, or control siRNA for 48 hours. A, B Transfected EC were stimulated with 0.1 μg/ml HLA class II antibody or control mIgG for 15 min. A, B, C Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting withanti-FAK antibody for siRNA knockdown efficiency, or A, B with anti-phospho-Src Tyr418, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Vinculin, Src, PI3K, Akt, mTOR, S6K, S6RP, ERK, β-actin antibodies to confirm equal loading of proteins. C β The membrane was immunoblotted with anti-Vinculin, β-tubulin, or β-actin antibody for FAK siRNA knockdown specificity. D, E EC were detached, incubated with 1.0 μg/ml HLA class II antibody for 30 min at 4°C, washed 3 timesand incubated with FITC-conjugated goat anti-mouse F(ab’)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on a LSRFortessa flow cytometer and calculated using FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one way ANOVA with Fisher's LSD. F, G Starved cells were stimulated with 1.0 μg/ml HLA class II antibody for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percent of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. *p
    Figure Legend Snippet: Effect of FAK siRNA transfection on HLA II antibody-stimulated activation of signal transduction networks and cell proliferation A, D, F EC were infected with Ad-LacZ or Ad-CIITA or B, E, G pretreated with TNFα/IFNγ for 48 hours. A, B, C, D, E, F, G Starved EC were transfected with 100 nM of FAK, or control siRNA for 48 hours. A, B Transfected EC were stimulated with 0.1 μg/ml HLA class II antibody or control mIgG for 15 min. A, B, C Proteins in the pre-cleared cell lysates were separated by 6∼15% SDS-PAGE followed by immunoblotting withanti-FAK antibody for siRNA knockdown efficiency, or A, B with anti-phospho-Src Tyr418, p85 PI3K Tyr458, Akt Thr308, Akt Ser473, mTOR Ser2448, S6K Thr389, S6RP Ser240/244, or ERK Thr202/Tyr204. The membranes were re-probed with anti-Vinculin, Src, PI3K, Akt, mTOR, S6K, S6RP, ERK, β-actin antibodies to confirm equal loading of proteins. C β The membrane was immunoblotted with anti-Vinculin, β-tubulin, or β-actin antibody for FAK siRNA knockdown specificity. D, E EC were detached, incubated with 1.0 μg/ml HLA class II antibody for 30 min at 4°C, washed 3 timesand incubated with FITC-conjugated goat anti-mouse F(ab’)2 fragment for 30 min at 4°C. The fluorescence intensity was measured on a LSRFortessa flow cytometer and calculated using FACSDiva program. The bar graphs show the mean ± SEM of median channel fluorescence values of HLA-II expression on EC and were analyzed by one way ANOVA with Fisher's LSD. F, G Starved cells were stimulated with 1.0 μg/ml HLA class II antibody for 48 h, incorporated with BrdU for 2 h, and harvested. EC proliferation was measured by flow cytometry, DNA synthesis S phase was gated, and proliferation index is presented as fold increase in the percent of cells positive for BrdU normalized to negative control. The bar graphs show the mean ± SEM fold change of proliferation index. *p

    Techniques Used: Transfection, Activation Assay, Transduction, Infection, SDS Page, Incubation, Fluorescence, Flow Cytometry, Cytometry, Expressing, DNA Synthesis, Negative Control

    23) Product Images from "Loss of Cbl–PI3K Interaction Enhances Osteoclast Survival due to p21-Ras Mediated PI3K Activation Independent of Cbl-b"

    Article Title: Loss of Cbl–PI3K Interaction Enhances Osteoclast Survival due to p21-Ras Mediated PI3K Activation Independent of Cbl-b

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.24779

    Cbl but not Cbl-b modulates PI3K activity during RANK signaling in osteoclasts. OCLs were serum-starved for 90 min and then treated with RANKL (50 ng/ml) for the indicated times. A: Blots were probed with anti-phospho-ERK (Thr202/Tyr204) antibodies (top panel) or pIKKα/β (bottom panel) and then stripped and re-probed with total ERK or IKKα antibodies. B: OCLs were treated as described above and blots were probed with anti-phospho-AKT-Thr308 antibodies (top panel) or anti-phospho-GSKβ (bottom panel) then stripped and re-probed for total proteins. To correct for experimental variability, the amounts of total proteins in individual bands were quantified by using Odyssey Infrared Imaging Systems software 2.1 (LICOR Biosciences), and the ratio of phosphorylated protein to total protein is shown at the bottom of the panels. A representative experiment of four repetitions is shown.
    Figure Legend Snippet: Cbl but not Cbl-b modulates PI3K activity during RANK signaling in osteoclasts. OCLs were serum-starved for 90 min and then treated with RANKL (50 ng/ml) for the indicated times. A: Blots were probed with anti-phospho-ERK (Thr202/Tyr204) antibodies (top panel) or pIKKα/β (bottom panel) and then stripped and re-probed with total ERK or IKKα antibodies. B: OCLs were treated as described above and blots were probed with anti-phospho-AKT-Thr308 antibodies (top panel) or anti-phospho-GSKβ (bottom panel) then stripped and re-probed for total proteins. To correct for experimental variability, the amounts of total proteins in individual bands were quantified by using Odyssey Infrared Imaging Systems software 2.1 (LICOR Biosciences), and the ratio of phosphorylated protein to total protein is shown at the bottom of the panels. A representative experiment of four repetitions is shown.

    Techniques Used: Activity Assay, Imaging, Software

    24) Product Images from "Cholestane-3?, 5?, 6?-triol Suppresses Proliferation, Migration, and Invasion of Human Prostate Cancer Cells"

    Article Title: Cholestane-3?, 5?, 6?-triol Suppresses Proliferation, Migration, and Invasion of Human Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065734

    Effect of triol treatment on the abundance and phosphorylation status of signaling proteins. Protein expression of Akt1, phospho-Akt Thr308, phospho-Akt Ser473, PDK1, phospho-p44/42 MAPK Thr202/Tyr204, c-Myc, Skp2, p27 Kip1 , FASN, GAPDH, β-actin, and α-tubulin in CDXR-3, DU-145, and PC-3 cells treated with 0, 10 or 20 µM triol for 48 hrs were assayed by Western blotting. Values represent relative protein abundance.
    Figure Legend Snippet: Effect of triol treatment on the abundance and phosphorylation status of signaling proteins. Protein expression of Akt1, phospho-Akt Thr308, phospho-Akt Ser473, PDK1, phospho-p44/42 MAPK Thr202/Tyr204, c-Myc, Skp2, p27 Kip1 , FASN, GAPDH, β-actin, and α-tubulin in CDXR-3, DU-145, and PC-3 cells treated with 0, 10 or 20 µM triol for 48 hrs were assayed by Western blotting. Values represent relative protein abundance.

    Techniques Used: Expressing, Western Blot

    25) Product Images from "Increased postexercise insulin sensitivity is accompanied by increased AS160 phosphorylation in slow‐twitch soleus muscle"

    Article Title: Increased postexercise insulin sensitivity is accompanied by increased AS160 phosphorylation in slow‐twitch soleus muscle

    Journal: Physiological Reports

    doi: 10.14814/phy2.12162

    Phosphorylation of Akt, AS160, and TBC1D1 in rat soleus muscles immediately after treadmill exercise (180 min at 9 m/min). Details are provided in the text. Frozen muscles were used to measure the phosphorylation of Akt Ser473 (A), Akt Thr308 (B), AS160 Thr642 (C), AS160 Ser588 (D), TBC1D1 Thr590 (E), and TBC1D1 Ser237 (F). Values are mean ± SE ( n = 7–8). * P
    Figure Legend Snippet: Phosphorylation of Akt, AS160, and TBC1D1 in rat soleus muscles immediately after treadmill exercise (180 min at 9 m/min). Details are provided in the text. Frozen muscles were used to measure the phosphorylation of Akt Ser473 (A), Akt Thr308 (B), AS160 Thr642 (C), AS160 Ser588 (D), TBC1D1 Thr590 (E), and TBC1D1 Ser237 (F). Values are mean ± SE ( n = 7–8). * P

    Techniques Used:

    Phosphorylation of Akt, AS160, and TBC1D1 in rat soleus muscles 2 h after treadmill exercise (180 min at 9 m/min). See the Materials and Methods section for details. Frozen muscles were used to measure the phosphorylation of Akt Ser473 (A), Akt Thr308 (B), AS160 Thr642 (C), AS160 Ser588 (D), TBC1D1 Thr590 (E), and TBC1D1 Ser237 (F). Values are mean ± SE, (A–D) n = 7–8; (E) n = 6–7; (F) n = 7–8. † P
    Figure Legend Snippet: Phosphorylation of Akt, AS160, and TBC1D1 in rat soleus muscles 2 h after treadmill exercise (180 min at 9 m/min). See the Materials and Methods section for details. Frozen muscles were used to measure the phosphorylation of Akt Ser473 (A), Akt Thr308 (B), AS160 Thr642 (C), AS160 Ser588 (D), TBC1D1 Thr590 (E), and TBC1D1 Ser237 (F). Values are mean ± SE, (A–D) n = 7–8; (E) n = 6–7; (F) n = 7–8. † P

    Techniques Used:

    26) Product Images from "Selective ?2-Adrenoreceptor Stimulation Attenuates Myocardial Cell Death and Preserves Cardiac Function After Ischemia-Reperfusion Injury"

    Article Title: Selective ?2-Adrenoreceptor Stimulation Attenuates Myocardial Cell Death and Preserves Cardiac Function After Ischemia-Reperfusion Injury

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    doi: 10.1161/ATVBAHA.112.251769

    A , Representative immunoblots of Akt-P Thr308 , Akt-P ser473 , total Akt, and GAPDH in tissue samples taken from the left ventricle of sham-operated mice at 15 minutes after receiving either vehicle (VEH) or arformoterol (ARF) (1 μg/kg) via intracardiac
    Figure Legend Snippet: A , Representative immunoblots of Akt-P Thr308 , Akt-P ser473 , total Akt, and GAPDH in tissue samples taken from the left ventricle of sham-operated mice at 15 minutes after receiving either vehicle (VEH) or arformoterol (ARF) (1 μg/kg) via intracardiac

    Techniques Used: Western Blot, Mouse Assay

    27) Product Images from "Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1"

    Article Title: Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

    Journal: Oncotarget

    doi:

    Micro-Western Array image and heatmap of abundance and phosphorylation fold changes of signaling proteins in LNCaP 104-R1 cells treated with CAPE (A) LNCaP 104-R1 cells were treated with 0, 10, 20, 40 μM CAPE for 96 h. Micro-Western Arrays were performed to measure the changes in abundance and modification of total Akt, Akt1, Akt2, phospho-Akt Thr308, phospho-Akt Ser473, AR, Cdk2, cyclin A, cyclin D1, c-Myc, p21 Cip1 , p27 Kip1 , Skp2, p70S6 kinase, phospho-p70 S6 kinase Thr421/Ser424, p90RSK, phospho-p90RSK Ser380, Rb, phospho-Rb Ser807/811, PTEN, Bcl-2, fatty acid synthase (FAS), p53, phospho-p53 Ser392, phospho-p53 Ser6, phospho-p53 Ser33, phospho-p53 Ser46, GSK3α, GSK3β, phospho-GSK3α Ser21, phospho-Gsk3β Ser9, SGK, phospho-SGK Ser255/Thr241, CKI, CKIIα, mTOR, phospho-mTOR Ser2448, PDK1, phospho-PDK1 Ser241, phospho-mTOR ser2481, p38 MAPK, phospho-p38 MAPK Thr180/Tyr182, NF-κB p65, and NF-κB p50. Protein abundance of α-tubulin and β-actin was used as loading control. Red color and green color indicated 680 nM and 780 nM wavelength detected by Licor Odyssey scanner for rabbit antibodies and mouse antibodies, respectively. Blue arrows indicated the correct location of band for the detected proteins. (B) Proteins were organized in the y-axis of the heatmap based on time of maximal fold change amplitude. Green color indicated decrease of protein expression while red color indicated increase of protein expression under treatment of CAPE.
    Figure Legend Snippet: Micro-Western Array image and heatmap of abundance and phosphorylation fold changes of signaling proteins in LNCaP 104-R1 cells treated with CAPE (A) LNCaP 104-R1 cells were treated with 0, 10, 20, 40 μM CAPE for 96 h. Micro-Western Arrays were performed to measure the changes in abundance and modification of total Akt, Akt1, Akt2, phospho-Akt Thr308, phospho-Akt Ser473, AR, Cdk2, cyclin A, cyclin D1, c-Myc, p21 Cip1 , p27 Kip1 , Skp2, p70S6 kinase, phospho-p70 S6 kinase Thr421/Ser424, p90RSK, phospho-p90RSK Ser380, Rb, phospho-Rb Ser807/811, PTEN, Bcl-2, fatty acid synthase (FAS), p53, phospho-p53 Ser392, phospho-p53 Ser6, phospho-p53 Ser33, phospho-p53 Ser46, GSK3α, GSK3β, phospho-GSK3α Ser21, phospho-Gsk3β Ser9, SGK, phospho-SGK Ser255/Thr241, CKI, CKIIα, mTOR, phospho-mTOR Ser2448, PDK1, phospho-PDK1 Ser241, phospho-mTOR ser2481, p38 MAPK, phospho-p38 MAPK Thr180/Tyr182, NF-κB p65, and NF-κB p50. Protein abundance of α-tubulin and β-actin was used as loading control. Red color and green color indicated 680 nM and 780 nM wavelength detected by Licor Odyssey scanner for rabbit antibodies and mouse antibodies, respectively. Blue arrows indicated the correct location of band for the detected proteins. (B) Proteins were organized in the y-axis of the heatmap based on time of maximal fold change amplitude. Green color indicated decrease of protein expression while red color indicated increase of protein expression under treatment of CAPE.

    Techniques Used: Western Blot, Modification, Expressing

    CAPE treatment affected abundance and phosphorylation of proteins involved in PI3K-Akt signaling pathways (A) Protein expression of Akt, Akt1, Akt2, phospho-Akt Thr308, phospho-Akt Ser473, PDK1, phospho-PDK1 Ser241, SGK, phospho-SGK Ser255/Thr256, CREB, phospho-CREB Ser133, FAS, p70S6 kinase, phospho-p70 S6 kinase Thr421/Ser424, mTOR, phospho-mTOR Ser2448, phospho-mTOR Ser2481, GSK3α, phospho-GSK3α Ser21, GSK3β, phospho-GSK3β Ser9, Bcl-2, p38 MAPK, phospho-p38 MAPK Thr180/Tyr182, p90RSK, phospho-p90RSK S380, Bad, Bax, CKI, CKIIα, CKIIβ, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and TRIB3 in LNCaP 104-R1 cells treated with 0, 10, 20, and 40 μM CAPE for 96 h were assayed by Western blotting. Protein abundance of α-tubulin and β-actin was used as loading control. (B) Proteins expression level was organized in the y-axis of the heatmap based on time of maximal fold change amplitude. Green color indicated decrease of protein expression while red color indicated increase of protein expression under treatment of CAPE.
    Figure Legend Snippet: CAPE treatment affected abundance and phosphorylation of proteins involved in PI3K-Akt signaling pathways (A) Protein expression of Akt, Akt1, Akt2, phospho-Akt Thr308, phospho-Akt Ser473, PDK1, phospho-PDK1 Ser241, SGK, phospho-SGK Ser255/Thr256, CREB, phospho-CREB Ser133, FAS, p70S6 kinase, phospho-p70 S6 kinase Thr421/Ser424, mTOR, phospho-mTOR Ser2448, phospho-mTOR Ser2481, GSK3α, phospho-GSK3α Ser21, GSK3β, phospho-GSK3β Ser9, Bcl-2, p38 MAPK, phospho-p38 MAPK Thr180/Tyr182, p90RSK, phospho-p90RSK S380, Bad, Bax, CKI, CKIIα, CKIIβ, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and TRIB3 in LNCaP 104-R1 cells treated with 0, 10, 20, and 40 μM CAPE for 96 h were assayed by Western blotting. Protein abundance of α-tubulin and β-actin was used as loading control. (B) Proteins expression level was organized in the y-axis of the heatmap based on time of maximal fold change amplitude. Green color indicated decrease of protein expression while red color indicated increase of protein expression under treatment of CAPE.

    Techniques Used: Expressing, Western Blot

    28) Product Images from "Synergistic Antitumor Effect of Sorafenib in Combination with ATM Inhibitor in Hepatocellular Carcinoma Cells"

    Article Title: Synergistic Antitumor Effect of Sorafenib in Combination with ATM Inhibitor in Hepatocellular Carcinoma Cells

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.19033

    Sorafenib and KU-55933 inhibit cell proliferation and induce apoptosis by inactivating Akt signaling pathway in a synergistic manner. Both floating and attached cells were collected after treatment. Cells were lysed by TGN lysis buffer, and cell lysates were subjected to SDS-PAGE and immunoblotting. PARP, cleaved PARP, phospho-Akt at Thr308, phosph-mTOR, phospho-p70S6K, LC3A/B and β-actin were detected. A.B.C represent results of HepG2, Huh7 and Hep3B cell lines respectively treated by sorafenib (5 µmol/L) and KU-55933(10 µmol/L) alone or combination for 24h, β-actin was detected as a control. The results in A to C are representative of three individual experiments.
    Figure Legend Snippet: Sorafenib and KU-55933 inhibit cell proliferation and induce apoptosis by inactivating Akt signaling pathway in a synergistic manner. Both floating and attached cells were collected after treatment. Cells were lysed by TGN lysis buffer, and cell lysates were subjected to SDS-PAGE and immunoblotting. PARP, cleaved PARP, phospho-Akt at Thr308, phosph-mTOR, phospho-p70S6K, LC3A/B and β-actin were detected. A.B.C represent results of HepG2, Huh7 and Hep3B cell lines respectively treated by sorafenib (5 µmol/L) and KU-55933(10 µmol/L) alone or combination for 24h, β-actin was detected as a control. The results in A to C are representative of three individual experiments.

    Techniques Used: Lysis, SDS Page

    29) Product Images from "IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells"

    Article Title: IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2016.00626

    Effects of IL-15 signaling on mediators of glucose uptake and proteins downstream of IL-2βγ receptor signaling . Western blotting of (A) phospho-Akt-Thr308, TBCD1, TBCD4, and total Akt; (B) phospho-AMPK-Thr172 and total AMPK; (C) phospho-Jak1 and total Jak1; (D) phospho-Jak3 and total Jak3. Assessments were carried out on whole cell lysates from differentiated C2C12 myotubes following 24 h treatment with 100 ng/ml of IL-15. GAPDH was used as a loading control for western blotting. Vehicle cells were used as a control to calculate protein expression. Image J was used to quantify band density in western blotting experiments. All values are displayed as means ± SEM, n = 6–9 per group, * P
    Figure Legend Snippet: Effects of IL-15 signaling on mediators of glucose uptake and proteins downstream of IL-2βγ receptor signaling . Western blotting of (A) phospho-Akt-Thr308, TBCD1, TBCD4, and total Akt; (B) phospho-AMPK-Thr172 and total AMPK; (C) phospho-Jak1 and total Jak1; (D) phospho-Jak3 and total Jak3. Assessments were carried out on whole cell lysates from differentiated C2C12 myotubes following 24 h treatment with 100 ng/ml of IL-15. GAPDH was used as a loading control for western blotting. Vehicle cells were used as a control to calculate protein expression. Image J was used to quantify band density in western blotting experiments. All values are displayed as means ± SEM, n = 6–9 per group, * P

    Techniques Used: Western Blot, Expressing

    30) Product Images from "Caffeic Acid Phenethyl Ester Suppresses Proliferation and Survival of TW2.6 Human Oral Cancer Cells via Inhibition of Akt Signaling"

    Article Title: Caffeic Acid Phenethyl Ester Suppresses Proliferation and Survival of TW2.6 Human Oral Cancer Cells via Inhibition of Akt Signaling

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms14058801

    Effects of CAPE treatment on the abundance and phosphorylation status of signaling proteins. Protein expression of total Akt, Akt1, Akt2, Akt3, phospho-Akt Ser473, phospho-Akt Thr308, GSK3β, Rb, phospho-Rb Ser807/811, cyclin D1, and Skp2, FOXO1, FOXO3a, phospho-FOXO1 Thr24, phospho-FOXO3a Thr32, NF-κB, phospho-NF-κB Ser536, p27 Kip , and β-actin in TW2.6 cells treated with 0, 50, or 100 μM CAPE for 48 h were assayed by Western blotting. Experiments were repeated three times.
    Figure Legend Snippet: Effects of CAPE treatment on the abundance and phosphorylation status of signaling proteins. Protein expression of total Akt, Akt1, Akt2, Akt3, phospho-Akt Ser473, phospho-Akt Thr308, GSK3β, Rb, phospho-Rb Ser807/811, cyclin D1, and Skp2, FOXO1, FOXO3a, phospho-FOXO1 Thr24, phospho-FOXO3a Thr32, NF-κB, phospho-NF-κB Ser536, p27 Kip , and β-actin in TW2.6 cells treated with 0, 50, or 100 μM CAPE for 48 h were assayed by Western blotting. Experiments were repeated three times.

    Techniques Used: Expressing, Western Blot

    31) Product Images from "Lack of Neurodegeneration in Transgenic Mice Overexpressing Mutant Amyloid Precursor Protein Is Associated with Increased Levels of Transthyretin and the Activation of Cell Survival Pathways"

    Article Title: Lack of Neurodegeneration in Transgenic Mice Overexpressing Mutant Amyloid Precursor Protein Is Associated with Increased Levels of Transthyretin and the Activation of Cell Survival Pathways

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-17-07380.2002

    Activation of the IGF-1 receptor, Akt, and Erk1/2 in the hippocampus of APP Sw mice. A , Immunoprecipitation for phosphorylated tyrosine and subsequent immunoblotting for the β-subunit of the insulin-like growth factor-1 receptor ( IGF-1Rβ ) revealed an increase in tyrosine-phosphorylated IGF-1Rβ in APP Sw mice (+APP Sw ) when compared with nontransgenic controls (−APP Sw ) in the hippocampus but not in the cerebellum. B–G , Hippocampal neurons in CA1 stain faintly or not at all for Akt phosphorylated at Thr308 in nontransgenic control animals ( B ), but neurons in CA1 in APP Sw mice are phospho-Akt positive ( C, D ). Hippocampal neurons in the dentate gyrus (DG) are faintly positive for phospho-Akt in control mice ( E ), but this staining is increased in the DG of APP Sw mice ( F, G ). A hematoxylin counterstain of C and F reveals the laminar organization of the CA1 and DG neurons and the nuclear localization of phospho-Akt within these neurons in APP Sw mice ( D, G ). H–M , Hippocampal neurons in CA1 ( H ) or DG ( K ) in control mice do not show significant staining for Erk1 phosphorylated at Thr202 and Tyr204 or Erk2 phosphorylated at Thr183 and Tyr185. However, APP Sw mice do show positive staining for phospho-Erk1/2 in CA1 ( I, J ) and DG ( L, M ). A hematoxylin counterstain of I and L reveals the laminar organization of the CA1 and DG neurons and the nuclear localization of phopsho-Erk1/2 within these neurons in APP Sw mice ( J, M ). Scale bars: J (for H–J ), M (for B–G, K , L ), 10 μm.
    Figure Legend Snippet: Activation of the IGF-1 receptor, Akt, and Erk1/2 in the hippocampus of APP Sw mice. A , Immunoprecipitation for phosphorylated tyrosine and subsequent immunoblotting for the β-subunit of the insulin-like growth factor-1 receptor ( IGF-1Rβ ) revealed an increase in tyrosine-phosphorylated IGF-1Rβ in APP Sw mice (+APP Sw ) when compared with nontransgenic controls (−APP Sw ) in the hippocampus but not in the cerebellum. B–G , Hippocampal neurons in CA1 stain faintly or not at all for Akt phosphorylated at Thr308 in nontransgenic control animals ( B ), but neurons in CA1 in APP Sw mice are phospho-Akt positive ( C, D ). Hippocampal neurons in the dentate gyrus (DG) are faintly positive for phospho-Akt in control mice ( E ), but this staining is increased in the DG of APP Sw mice ( F, G ). A hematoxylin counterstain of C and F reveals the laminar organization of the CA1 and DG neurons and the nuclear localization of phospho-Akt within these neurons in APP Sw mice ( D, G ). H–M , Hippocampal neurons in CA1 ( H ) or DG ( K ) in control mice do not show significant staining for Erk1 phosphorylated at Thr202 and Tyr204 or Erk2 phosphorylated at Thr183 and Tyr185. However, APP Sw mice do show positive staining for phospho-Erk1/2 in CA1 ( I, J ) and DG ( L, M ). A hematoxylin counterstain of I and L reveals the laminar organization of the CA1 and DG neurons and the nuclear localization of phopsho-Erk1/2 within these neurons in APP Sw mice ( J, M ). Scale bars: J (for H–J ), M (for B–G, K , L ), 10 μm.

    Techniques Used: Activation Assay, Mouse Assay, Immunoprecipitation, Staining

    Related Articles

    Modification:

    Article Title: Effects and Interaction of Icariin, Curculigoside, and Berberine in Er-Xian Decoction, a Traditional Chinese Medicinal Formula, on Osteoclastic Bone Resorption
    Article Snippet: .. Reagents Reagents used in this study included α -modified minimum essential medium (α -MEM) and fetal bovine serum (FBS) (Gibco, US); 1, 25-dihydroxyvitamin D3 , dexamethasone, trypsin, and coomassie brilliant blue G-250 (Sigma, US); recombinant rat M-CSF (400-28) and RANKL (400-30) (Peprotech EC, US); OPG (sc11383), RANKL (sc9073), and β -actin (sc81178) antibody (Santa Cruz, USA); anti-phospho-Akt (9275), anti-phospho-ERK (3371), anti-phospho-JNK (9251), anti-phospho-p38 (9211), and anti-phospho-I-κ B (9245) (Cell Signaling Technology, Beverly, MA); NucBuster Protein Extraction kit (Merck, Novagen, Germany); Cathepsin K activity kit (Biovision, USA); pararosaniline, Hoechst 33258, and rhodamine-conjugated phalloidin (Sigma, USA). ..

    Western Blot:

    Article Title: GSNOR modulates hyperhomocysteinemia-induced T cell activation and atherosclerosis by switching Akt S-nitrosylation to phosphorylation
    Article Snippet: .. Antibodies for western blot analysis included anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), anti-Akt, anti-β-actin, anti-GAPDH, anti-flag (1:1000, all from Cell Signaling Technology, Danvers, MA, USA), anti-SNO-Cys (1:1000, Sigma-Aldrich, St. Louis, MO, USA), anti-iNOS (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Trx1 (1:1000, ABclonal Biotechnology, Hubei, China), and anti-GSNOR (1:1000, Proteintech, Rosemont, USA). .. Cells and aortas were collected, and total RNA was extracted by the TRIzol reagent method (Invitrogen, Carlsbad, CA, USA), then reverse-transcribed to cDNA and amplified using the AMV Reverse Transcription System (Promega, Madison, WI, USA).

    Protein Extraction:

    Article Title: Effects and Interaction of Icariin, Curculigoside, and Berberine in Er-Xian Decoction, a Traditional Chinese Medicinal Formula, on Osteoclastic Bone Resorption
    Article Snippet: .. Reagents Reagents used in this study included α -modified minimum essential medium (α -MEM) and fetal bovine serum (FBS) (Gibco, US); 1, 25-dihydroxyvitamin D3 , dexamethasone, trypsin, and coomassie brilliant blue G-250 (Sigma, US); recombinant rat M-CSF (400-28) and RANKL (400-30) (Peprotech EC, US); OPG (sc11383), RANKL (sc9073), and β -actin (sc81178) antibody (Santa Cruz, USA); anti-phospho-Akt (9275), anti-phospho-ERK (3371), anti-phospho-JNK (9251), anti-phospho-p38 (9211), and anti-phospho-I-κ B (9245) (Cell Signaling Technology, Beverly, MA); NucBuster Protein Extraction kit (Merck, Novagen, Germany); Cathepsin K activity kit (Biovision, USA); pararosaniline, Hoechst 33258, and rhodamine-conjugated phalloidin (Sigma, USA). ..

    Recombinant:

    Article Title: Effects and Interaction of Icariin, Curculigoside, and Berberine in Er-Xian Decoction, a Traditional Chinese Medicinal Formula, on Osteoclastic Bone Resorption
    Article Snippet: .. Reagents Reagents used in this study included α -modified minimum essential medium (α -MEM) and fetal bovine serum (FBS) (Gibco, US); 1, 25-dihydroxyvitamin D3 , dexamethasone, trypsin, and coomassie brilliant blue G-250 (Sigma, US); recombinant rat M-CSF (400-28) and RANKL (400-30) (Peprotech EC, US); OPG (sc11383), RANKL (sc9073), and β -actin (sc81178) antibody (Santa Cruz, USA); anti-phospho-Akt (9275), anti-phospho-ERK (3371), anti-phospho-JNK (9251), anti-phospho-p38 (9211), and anti-phospho-I-κ B (9245) (Cell Signaling Technology, Beverly, MA); NucBuster Protein Extraction kit (Merck, Novagen, Germany); Cathepsin K activity kit (Biovision, USA); pararosaniline, Hoechst 33258, and rhodamine-conjugated phalloidin (Sigma, USA). ..

    Activity Assay:

    Article Title: Effects and Interaction of Icariin, Curculigoside, and Berberine in Er-Xian Decoction, a Traditional Chinese Medicinal Formula, on Osteoclastic Bone Resorption
    Article Snippet: .. Reagents Reagents used in this study included α -modified minimum essential medium (α -MEM) and fetal bovine serum (FBS) (Gibco, US); 1, 25-dihydroxyvitamin D3 , dexamethasone, trypsin, and coomassie brilliant blue G-250 (Sigma, US); recombinant rat M-CSF (400-28) and RANKL (400-30) (Peprotech EC, US); OPG (sc11383), RANKL (sc9073), and β -actin (sc81178) antibody (Santa Cruz, USA); anti-phospho-Akt (9275), anti-phospho-ERK (3371), anti-phospho-JNK (9251), anti-phospho-p38 (9211), and anti-phospho-I-κ B (9245) (Cell Signaling Technology, Beverly, MA); NucBuster Protein Extraction kit (Merck, Novagen, Germany); Cathepsin K activity kit (Biovision, USA); pararosaniline, Hoechst 33258, and rhodamine-conjugated phalloidin (Sigma, USA). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Cell Signaling Technology Inc α phospho akt thr308
    LB-100 inhibits PP2A enzymatic activity and activates the mTORC1 pathway. CD3 T cells were isolated from mice splenocytes and cultured with or without stimulation using immobilized anti-CD3 (10 μg ml −1 ) and soluble anti-CD28 (2 μg ml −1 ). a PP2A enzymatic activity was measured after 3 h of activation. PP2A activity was measured as relative to activated control in presence of LB-100 dose titration. b Flow cytometry analyzing <t>AKT</t> phosphorylated at <t>Thr308</t> (p-AKT(T308)) or Ser473 (p-AKT(S473)) after 3 h of stimulation in presence of LB-100 dose titration. c Flow cytometry analyzing phosphorylated S6 (p-S6) in presence of LB-100 dose titration. * P
    α Phospho Akt Thr308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 2369 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α phospho akt thr308/product/Cell Signaling Technology Inc
    Average 90 stars, based on 2369 article reviews
    Price from $9.99 to $1999.99
    α phospho akt thr308 - by Bioz Stars, 2020-11
    90/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc rabbit anti phospho akt ser473
    HDL stimulated macrophage migration involves Rho kinase and <t>PI3K-Akt</t> 1 signaling. A. RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. B. RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either <t>phospho-Ser473-</t> or total-Akt. C. MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P
    Rabbit Anti Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho akt ser473/product/Cell Signaling Technology Inc
    Average 99 stars, based on 76 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho akt ser473 - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    Image Search Results


    LB-100 inhibits PP2A enzymatic activity and activates the mTORC1 pathway. CD3 T cells were isolated from mice splenocytes and cultured with or without stimulation using immobilized anti-CD3 (10 μg ml −1 ) and soluble anti-CD28 (2 μg ml −1 ). a PP2A enzymatic activity was measured after 3 h of activation. PP2A activity was measured as relative to activated control in presence of LB-100 dose titration. b Flow cytometry analyzing AKT phosphorylated at Thr308 (p-AKT(T308)) or Ser473 (p-AKT(S473)) after 3 h of stimulation in presence of LB-100 dose titration. c Flow cytometry analyzing phosphorylated S6 (p-S6) in presence of LB-100 dose titration. * P

    Journal: Nature Communications

    Article Title: Pharmacologic inhibition of protein phosphatase-2A achieves durable immune-mediated antitumor activity when combined with PD-1 blockade

    doi: 10.1038/s41467-018-04425-z

    Figure Lengend Snippet: LB-100 inhibits PP2A enzymatic activity and activates the mTORC1 pathway. CD3 T cells were isolated from mice splenocytes and cultured with or without stimulation using immobilized anti-CD3 (10 μg ml −1 ) and soluble anti-CD28 (2 μg ml −1 ). a PP2A enzymatic activity was measured after 3 h of activation. PP2A activity was measured as relative to activated control in presence of LB-100 dose titration. b Flow cytometry analyzing AKT phosphorylated at Thr308 (p-AKT(T308)) or Ser473 (p-AKT(S473)) after 3 h of stimulation in presence of LB-100 dose titration. c Flow cytometry analyzing phosphorylated S6 (p-S6) in presence of LB-100 dose titration. * P

    Article Snippet: Anti-human: α-CD4 (A161A1, Biolegend, 2.5 μg ml−1 ), α-T-bet (4B10, Biolegend, 2.5 μg ml−1 ), α-Phospho-Akt (Ser473) (D9E, Cell Signaling, 0.5 μg ml−1 ), α-Phospho-Akt (Thr308) (D25E6, Cell Signaling, 0.5 μg ml−1 ), α-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, Cell Signaling, 0.5 μg ml−1 ).

    Techniques: Activity Assay, Isolation, Mouse Assay, Cell Culture, Activation Assay, Titration, Flow Cytometry, Cytometry

    Representative blots of nuclear FoxO3a ( A ), SIRT1 ( B ), phospho-Akt (Thr473), Akt ( C ), phospho-FoxO3a (Ser253), and total FoxO3a ( D ) in m/m and db/db mice with or without oligonol (OLG) supplement are shown ( n = 5–6/group). Significance ( p

    Journal: Nutrients

    Article Title: Oligonol, a Low-Molecular Weight Polyphenol Derived from Lychee, Alleviates Muscle Loss in Diabetes by Suppressing Atrogin-1 and MuRF1

    doi: 10.3390/nu9091040

    Figure Lengend Snippet: Representative blots of nuclear FoxO3a ( A ), SIRT1 ( B ), phospho-Akt (Thr473), Akt ( C ), phospho-FoxO3a (Ser253), and total FoxO3a ( D ) in m/m and db/db mice with or without oligonol (OLG) supplement are shown ( n = 5–6/group). Significance ( p

    Article Snippet: Akt, phospho-Akt (Thr308), FoxO3a, phospho-FoxO3a (Ser253), AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172) were purchased from Cell Signaling (Danvers, MA, USA).

    Techniques: Mouse Assay

    HDL stimulated macrophage migration involves Rho kinase and PI3K-Akt 1 signaling. A. RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. B. RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either phospho-Ser473- or total-Akt. C. MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P

    Journal: PLoS ONE

    Article Title: High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B, Type I, PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

    doi: 10.1371/journal.pone.0106487

    Figure Lengend Snippet: HDL stimulated macrophage migration involves Rho kinase and PI3K-Akt 1 signaling. A. RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. B. RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either phospho-Ser473- or total-Akt. C. MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P

    Article Snippet: After boiling, the samples were subjected to SDS-PAGE followed by immunoblotting with rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Cell Culture, Incubation, SDS Page, Mouse Assay

    SphK1 silencing downregulates Akt/mTOR signaling pathway in VHL -defective A498 and 786-O ccRCC cells. A498 and 786-O cells were untransfected or transfected with 20 nmol/l of siSphK1 or siScr for 72 h before the experiments followed by 4 h under normoxic or hypoxic condition. Cell lysates were assayed for Ser2448 phosphorylated mTOR (p-mTOR Ser2448) and mTOR expression ( a ); Thr389 phosphorylated p70S6K (p-p70S6K Thr389), Thr421/Ser424 phosphorylated p70S6K (p-p70S6K Thr421/Ser424) and p70S6K expression ( b ); Ser65 phosphorylated 4E-BP1 (p-4E-BP1 Ser65) and 4E-BP1 expression ( c ); Ser473 phosphorylated Akt (P-Akt Ser473) and Akt expression ( d ) were analyzed by immunoblotting. Similar results were obtained in three independent experiments.

    Journal: Oncogenesis

    Article Title: Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer

    doi: 10.1038/oncsis.2016.13

    Figure Lengend Snippet: SphK1 silencing downregulates Akt/mTOR signaling pathway in VHL -defective A498 and 786-O ccRCC cells. A498 and 786-O cells were untransfected or transfected with 20 nmol/l of siSphK1 or siScr for 72 h before the experiments followed by 4 h under normoxic or hypoxic condition. Cell lysates were assayed for Ser2448 phosphorylated mTOR (p-mTOR Ser2448) and mTOR expression ( a ); Thr389 phosphorylated p70S6K (p-p70S6K Thr389), Thr421/Ser424 phosphorylated p70S6K (p-p70S6K Thr421/Ser424) and p70S6K expression ( b ); Ser65 phosphorylated 4E-BP1 (p-4E-BP1 Ser65) and 4E-BP1 expression ( c ); Ser473 phosphorylated Akt (P-Akt Ser473) and Akt expression ( d ) were analyzed by immunoblotting. Similar results were obtained in three independent experiments.

    Article Snippet: Western-blot analysis and antibodies Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies.

    Techniques: Transfection, Expressing