Journal: PLoS ONE

Article Title: Regulation of mTORC1 Signaling by pH

doi: 10.1371/journal.pone.0021549

Figure Lengend Snippet: Rapid and reversible inhibition of mTORC1 signaling by acidic extracellular pH. A, MCF-7 cells were exposed for 5 min or 30 min to cell culture medium buffered to the indicated pH values. Lysates were analysed for mTORC1 activation by western blotting using antibodies to phospho-Thr 389 S6K and to S6K and for mTORC2 activation using antibodies to phospho-Ser 473 PKB/Akt and to PKB/Akt. Tubulin immunodetection was used as a protein loading control. B, MCF-7 cells grown in normal cell culture medium were exposed to medium buffered to pH 6.4 or 7.4 for 30 min. The medium was removed and replaced with medium buffered to pH 7.4 and samples were harvested immediately (0 min) or at the indicated times. In parallel, cells were exposed or not to the mTORC1 inhibitor rapamycin (Rapa, 30 nM) for 30 min in normal cell culture medium (CM). Lysates were analysed by immunoblotting. Note that changing the cell culture medium caused a minor and transient decrease in PKB/Akt Ser 473 phosphorylation that was independent of acidic pH. Results shown in all figures are representative of three or more independent experiments.

Article Snippet: Antibodies were from the following vendors: Cell Signaling Technology, phospho Thr389 S6K (#9205), phospho Ser473 PKB/Akt (#9271), PKB/Akt (#9272), phospho Ser 380 p90RSK (#9341), phospho Ser217/221 MEK1/2 (#9121), phospho Thr308 PKB/Akt (#9275), phospho Ser259 Raf-1 (#9421), phospho-p44/42 Thr202/204 MAPK (Erk1/2) (#9106), AMPK (#2603), phospho Thr172 AMPK (#2535), mTOR (#2972); Santa Cruz Biotechnology, Inc., S6K C-18 (#230), phospho Erk (#9106), Raf-1 E-10 (sc-7267), β-tubulin H-235 (#9104), Erk; Millipore, phospho Ser338 Raf-1(05-538); Upstate, 4G10 phosphotyrosine (Tyr(P)); Developmental Studies Hybridoma Bank, LAMP1 (H4A3); Invitrogen, Alexa fluor 488 goat-α-rabbit (#A11008) and Alexa fluor 568 goat-α-mouse (#A11004).

Techniques: Inhibition, Cell Culture, Activation Assay, Western Blot, Immunodetection

Journal: PLoS ONE

Article Title: Adipocyte-Specific Protein Tyrosine Phosphatase 1B Deletion Increases Lipogenesis, Adipocyte Cell Size and Is a Minor Regulator of Glucose Homeostasis

doi: 10.1371/journal.pone.0032700

Figure Lengend Snippet: Reduced in vivo insulin signaling in epididymal white adipose tissue from HFD-fed adip-crePTP1B −/− mice. A : Epididymal white adipose tissue immunoblots of insulin signaling components in chow- and HFD-fed fl/fl and adip-crePTP1B −/− (KO) and fl/fl (FL) mice after injection with saline or insulin (10 mU/kg). B : PTP1B levels and deletion efficiency of fl/fl ( n = 4–5) and adip-crePTP1B −/− mice ( n = 4) in epididymal white adipose tissue under chow- and HFD-fed conditions. Graphs C to F show phosphorylation levels of the indicated proteins in epididymal white adipose tissue after saline or insulin (10 mU/kg) injection of chow- or HFD-fed fl/fl and adip-crePTP1B −/− mice, as indicated. Phosphorylated proteins were normalized as shown in the graphs. C : IP: IR (IB: pY). D : IR Y1158. E : IRS-1 Y608. F : Akt/PKB S473. White bar = fl/fl; black bar = adip-crePTP1B −/− . Data are represented as mean ± SEM; data were analyzed using two-way ANOVA with Bonferroni multiple comparisons post-tests to compare between diets, and two-tailed Student's t test to compare between different genotypes on the same diet (* P

Article Snippet: Immunoblots were performed using antibodies from Cell Signaling (Cell Signaling by NEB, Hitchin, UK) (unless stated otherwise) against pIR Y1158, pIRS-1 S636/639, total IRS-1, pAkt/PKB S473, total Akt/PKB, pERK1/2 MAPK T202/Y204, pS6 ribosomal protein S235/236, pS6 ribosomal protein S240/244, p-p70S6K T389, total p70S6K, p-mTOR S2448, total mTOR (Santa Cruz, Insight Biotechnology, Wembley, UK) pGSK-3α S21, pGSK-3β S9, pAMPK T172, total AMPKα, pIR Y1162/63 (Invitrogen), total IR (Santa Cruz), SHP2 (Santa Cruz), pIRS-1 Y608 (CalBiochem), RBP4 (Dako, Cambridgeshire, UK), TC-PTP (R & D Systems) and PTP1B (Millipore, Chandlers Ford, UK).

Techniques: In Vivo, Mouse Assay, Western Blot, Injection, Two Tailed Test

Journal: PLoS ONE

Article Title: Adipocyte-Specific Protein Tyrosine Phosphatase 1B Deletion Increases Lipogenesis, Adipocyte Cell Size and Is a Minor Regulator of Glucose Homeostasis

doi: 10.1371/journal.pone.0032700

Figure Lengend Snippet: Reduced in vivo insulin signaling in subcutaneous white adipose tissue from HFD-fed adip-crePTP1B −/ − mice. A : Subcutaneous white adipose tissue immunoblots of insulin signaling components in chow- and HFD-fed fl/fl and adip-crePTP1B −/− (KO) and fl/fl (FL) mice after injection with saline or insulin (10 mU/kg). B : Akt/PKB S473 phosphorylation levels normalized to total Akt/PKB in subcutaneous white adipose tissue after saline or insulin (10 mU/kg) injection of chow- or HFD-fed fl/fl (FL) and adip-crePTP1B −/− mice (KO). C : Brown adipose tissue immunoblots of insulin signaling components in chow- and HFD-fed fl/fl and adip-crePTP1B −/− (KO) and fl/fl (FL) mice after injection with saline or insulin (10 mU/kg). White bar = fl/fl; black bar = adip-crePTP1B −/− . Data are represented as mean ± SEM; data were analyzed using two-tailed Student's t test (* P

Article Snippet: Immunoblots were performed using antibodies from Cell Signaling (Cell Signaling by NEB, Hitchin, UK) (unless stated otherwise) against pIR Y1158, pIRS-1 S636/639, total IRS-1, pAkt/PKB S473, total Akt/PKB, pERK1/2 MAPK T202/Y204, pS6 ribosomal protein S235/236, pS6 ribosomal protein S240/244, p-p70S6K T389, total p70S6K, p-mTOR S2448, total mTOR (Santa Cruz, Insight Biotechnology, Wembley, UK) pGSK-3α S21, pGSK-3β S9, pAMPK T172, total AMPKα, pIR Y1162/63 (Invitrogen), total IR (Santa Cruz), SHP2 (Santa Cruz), pIRS-1 Y608 (CalBiochem), RBP4 (Dako, Cambridgeshire, UK), TC-PTP (R & D Systems) and PTP1B (Millipore, Chandlers Ford, UK).

Techniques: In Vivo, Mouse Assay, Western Blot, Injection, Two Tailed Test

Journal: Cardiovascular Diabetology

Article Title: Diacylglycerol kinase ? inhibits myocardial atrophy and restores cardiac dysfunction in streptozotocin-induced diabetes mellitus

doi: 10.1186/1475-2840-7-2

Figure Lengend Snippet: Representative immunoblots of left ventricular extracts with anti- phosphospecific Akt/PKB antibody (upper gel) in WT and DGKζ-TG mice at 8 weeks after injection of STZ or citrate buffer solution. The abundance of Akt protein was demonstrated by immunoblots with an antibody to total Akt (lower gel). Densitometric analyses of Akt phosphorylation were performed using 8 mice for each group. *P

Article Snippet: To examine phosphorylation activity of Akt/PKB, Western blotting was performed with an anti-phosphospecific Akt (Ser473) antibody, which detects Akt only when phosphorylated at Ser473 (Cell Signalling, Denvers, MA, USA) as reported previously [ ].

Techniques: Western Blot, Mouse Assay, Injection

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: PKB (PKB/Akt) mediates albumin uptake in proximal tubule cells. HKC-8 cells are transfected with plasmids encoding wild-type (WT) and constitutively active (CA)-PKB/Akt. Cells were treated with 100 μg/ml of FITC-albumin for 1 h. Albumin uptake

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Transfection

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Albumin overload results in downregulation of megalin, Dab2, and PKB/Akt in association with proximal tubule cell apoptosis. Coimmunoprecipitation experiments were performed after HKC-8 cells were treated with 10 mg/ml of albumin for 24 h. Different dilutions

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Lack of interaction between megalin and PKB/Akt. There is a lack of coimmunoprecipitation between megalin and PKB/Akt in HKC-8 and HeLa cells transfected with minimegalin ( A–B ). Cells were transfected with a HA-tagged chimeric cDNA consisting

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Transfection

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Nonselective PKB/Akt inhibitor causes concentration-dependent decrease in albumin uptake. HKC-8 cells were pretreated with different concentrations of API-2, a specific PKB/Akt inhibitor, for 24 h before incubation with 100 μg/ml of albumin. API-2

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Concentration Assay, Incubation

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Expression of PKB/Akt has a direct effect on Dab2. Overexpression of PKB/Akt with WT and CA-PKB/Akt causes an increase in Dab2 expression suggesting that Dab2 is downstream from PKB/Akt. HKC-8 cells were treated with 100 μg/ml of albumin after

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Expressing, Over Expression

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Expression of PKB/Akt has a direct effect on Dab2. Transfection of HKC-8 cells with dominant negative PKB/Akt caused downregulation of Dab2 expression confirming the role of PKB/Akt on stabilization of Dab2. Human kidney proximal tubule cells were treated

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Expressing, Transfection, Dominant Negative Mutation

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: PKB/Akt regulates albumin uptake via its interaction with Dab2. Silencing Dab2 by interfering RNA decreased albumin uptake in human proximal tubule cells confirming the role of Dab2 in albumin endocytosis in proximal tubule cells. Double transfection

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Transfection

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Interaction between disabled 2 (Dab2) and PKB/Akt. Coimmunoprecipitation experiments demonstrated an interaction between Dab2 and PKB/Akt. Immunofluorescence staining showed colocalization of Dab2 and PKB/Akt in HKC-8 cells. The immunofluorescence of

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Immunofluorescence, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: PKB/Akt binds to proline-rich domain (PRD) of Dab2. Schematic illustration of domain organization of Dab2 and various GST-Dab2 constructs used in the pull-down experiments ( A ). Approximately 100 μg of GST, GST-Dab2 (1–206), GST-Dab2 (1–368),

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Construct

Journal: American Journal of Physiology - Renal Physiology

Article Title: PKB/Akt partners with Dab2 in albumin endocytosis

doi: 10.1152/ajprenal.00289.2011

Figure Lengend Snippet: Overexpression of PKB/Akt with CA-PKB/Akt increased albumin uptake. HKC-8 cells were incubated with increasing concentrations of albumin (100 μg/ml, 500 μg/ml, 1 mg/ml) after transfection with CA-PKB. Cell lysates were blotted with an

Article Snippet: These findings suggested the lack of a strong interaction between megalin and PKB/Akt.

Techniques: Over Expression, Incubation, Transfection

Journal: Genes & Development

Article Title: Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation

doi: 10.1101/gad.255603

Figure Lengend Snippet: Effect of ILK deletion on proliferation and PKB/Akt and GSK-3β phosphorylation in fibroblasts treated with insulin or PDGF. ( A ) ILK-floxed (fl/fl) and ILK -null (−/−) cells were lysed with RIPA buffer, and total cell lysates were resolved in SDS gel, transferred to a PDVF membrane, and probed with anti-cyclin D1/2, anti-cyclin A, and anti-α-tubulin. The band intensities were measured by densitometry, and the intensity of the first ILK fl/fl sample value was set to 1. Each value is shown relative to this control value. ( B ) BrdU incorporation in ILK-floxed (fl/fl) and ILK -null (−/−) cells. The cells were incorporated in the presence of BrdU for 2 h. BrdU incorporation was determined by colorimetric immunoassays. Bars represent the mean of BrdU incorporation quantified by spectrophotometric analysis at OD = 492 nm. Error bars represent ±S.D. (n = 8). ( C ) ILK-floxed (fl/fl) and ILK-deficient (−/−) cells were serum-starved and either untreated (cont.) or treated with insulin (Ins) or PDGF for 15 or 45 min. Total lysates of the cells were resolved in SDS gel, transferred to a PDVF membrane, and probed with anti-PKB/Akt, anti-phospho-PKB/Akt (Thr 308 or Ser 473), anti-GSK-3β, anti-phosphoGSK-3β (Ser 9), anti-cyclin D1/2, anti-cyclin A, and anti-α-tubulin.

Article Snippet: The following antibodies were used for the analyses: mouse monoclonal antibodies against ILK (clone 3, Transduction lab; clone 65.1.9, Upstate); rabbit antibody against ILK (Upstate); rat mAb against E-cadherin (Zymed); rat mAb against platelet endothelial cell adhesion molecule (PECAM; Pharmingen); rabbit antibody against neurofilament M (Chemicon); mouse mAb against myosin (clone MF22); rabbit antibody against laminin-γ1; mouse mAb against vinculin (sigma); mouse mAb against paxillin (Transduction lab); rabbit antibody against integrin β1 ( ); mouse mAb against α smooth muscle cell actin (Sigma); rabbit antibodies against PKB/Akt and phospho-PKB/Akt (Thr 308, Ser 473; Cell Signaling Technology); mouse mAb against GSK-3β (Transduction lab); rabbit antibody against phospho-GSK-3β (Ser 9; Biosource Int.); mouse mAb against cyclin D1/2 (Upstate); rabbit antibody against cyclin A (Santa Cruz Biotechnology); and rat mAb against α-tubulin.

Techniques: SDS-Gel, BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Metabolic Outcome of Female Mice Exposed to a Mixture of Low-Dose Pollutants in a Diet-Induced Obesity Model

doi: 10.1371/journal.pone.0124015

Figure Lengend Snippet: Western blot analysis of the ratio between phosphorylated PKB/AKT versus total PKB/AKT in the gastrocnemius muscle removed from F1-HF0 and F1-HFp mice stimulated or not by insulin (the fold stimulation by insulin, relative to the mean of the corresponding control without insulin was assigned above each band shown). For quantification, each blot has been hybridized to α-tubulin and the graphs below represent means ± SE; *p

Article Snippet: Immunoblotting was performed using rabbit polyclonal antibodies directed against ERα (sc-542, Santa Cruz Biotechnology, CliniSciences, Nanterre, France), EST (sc-292049, Santa Cruz Biotechnology), phospho-AKT/PKB (Ser473) (#4060, Cell Signaling Technology Europe, Leiden, The Nederlands) and total AKT/PKB (#9272, Cell Signaling Technology Europe), or mouse monoclonal antibodies directed against α-tubulin (sc-5286, Santa Cruz Biotechnology).

Techniques: Western Blot, Mouse Assay

Journal: PLoS ONE

Article Title: Metabolic Outcome of Female Mice Exposed to a Mixture of Low-Dose Pollutants in a Diet-Induced Obesity Model

doi: 10.1371/journal.pone.0124015

Figure Lengend Snippet: (A) Effect of the mixture of pollutants on hepatic mRNA expression of xenobiotic nuclear receptors; (B) Analysis by Western blot of the ratio between phosphorylated PKB/AKT versus total PKB/AKT in liver removed from F1-HF0 and F1-HFp mice stimulated or not by insulin. For quantification, each blot has been hybridized to α-tubulin and the graphs below represent means ± SE; n = 7–8 mice. (C) and (D) Hepatic expression of Nr1c1 and of several PPARα-regulated genes (C) and of genes encoding proteins involved in lipogenesis (D). Results are means ± SE; *p

Article Snippet: Immunoblotting was performed using rabbit polyclonal antibodies directed against ERα (sc-542, Santa Cruz Biotechnology, CliniSciences, Nanterre, France), EST (sc-292049, Santa Cruz Biotechnology), phospho-AKT/PKB (Ser473) (#4060, Cell Signaling Technology Europe, Leiden, The Nederlands) and total AKT/PKB (#9272, Cell Signaling Technology Europe), or mouse monoclonal antibodies directed against α-tubulin (sc-5286, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Mouse Assay

Journal: BMC Developmental Biology

Article Title: Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3??

doi: 10.1186/1471-213X-10-73

Figure Lengend Snippet: Specific increase in PKBβ/Akt2 is detected in Irs2-/- kidney . (A) Twenty μg of protein lysates from Irs2 +/+ (n = 3), Irs2 +/- (n = 3) and Irs2 -/- (n = 6) mice were separated on 10% SDS-PAGE and probed by Western blotting with anti-PKBα/Akt1, PKBβ/Akt2, PKBγ/Akt3 and anti-PKB/Akt antibodies. GAPDH was used as a loading control. (B) Band intensities were quantified using Scion Image software. The intensity ratio of anti-PKBβ/GAPDH is shown. ** p

Article Snippet: Antibodies at the following dilutions were used: IRS-2 (1:500 generated in-house, Prof. Morris White), IRS-2 (Millipore, used for immunoprecipitation), polyclonal pAkt Ser473 (1:1000, Cell Signaling), pAkt Thr 308 (1:1000, Cell Signaling), pGSK-3β Ser9 (1:1000, Cell Signaling), total GSK-3β (1:1000, Cell Signaling), β-catenin (1:1000, BD Biosciences), total YAP (1:1000, Cell Signaling), pYAP Ser127 (1:1000, Cell Signaling), isoform specific PKB/Akt antibodies PKBα (1:500), PKBβ (1:500), PKBγ (1:5000) were a generous gift from Dr. Brian Hemmings, Friedrich Miescher Institute, Basel, Switzerland, total PKB/Akt (1:1000, Cell Signaling), GAPDH (1:5000, Cell Signaling), β-actin (1:25,000, Sigma).

Techniques: Mouse Assay, SDS Page, Western Blot, Software

Journal: BMC Developmental Biology

Article Title: Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3??

doi: 10.1186/1471-213X-10-73

Figure Lengend Snippet: Increased PKB/Akt expression in Irs2 -/- mouse kidney . Twenty μg of kidney protein lysates from 5-6 wk Irs2 +/+ , Irs2 +/- and Irs2 -/- 5-6 wk male (A) and female (B) groups were separated on 10% SDS-PAGE and probed by Western blotting with anti-PKB/Akt. GAPDH was used as a loading control. Band intensities were calculated using Scion Image software. The intensity ratios of PKB/GAPDH are shown. ** p

Article Snippet: Antibodies at the following dilutions were used: IRS-2 (1:500 generated in-house, Prof. Morris White), IRS-2 (Millipore, used for immunoprecipitation), polyclonal pAkt Ser473 (1:1000, Cell Signaling), pAkt Thr 308 (1:1000, Cell Signaling), pGSK-3β Ser9 (1:1000, Cell Signaling), total GSK-3β (1:1000, Cell Signaling), β-catenin (1:1000, BD Biosciences), total YAP (1:1000, Cell Signaling), pYAP Ser127 (1:1000, Cell Signaling), isoform specific PKB/Akt antibodies PKBα (1:500), PKBβ (1:500), PKBγ (1:5000) were a generous gift from Dr. Brian Hemmings, Friedrich Miescher Institute, Basel, Switzerland, total PKB/Akt (1:1000, Cell Signaling), GAPDH (1:5000, Cell Signaling), β-actin (1:25,000, Sigma).

Techniques: Expressing, SDS Page, Western Blot, Software

Journal: BMC Developmental Biology

Article Title: Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3??

doi: 10.1186/1471-213X-10-73

Figure Lengend Snippet: Increased insulin-stimulated PKB/Akt activation in Irs2-/- kidney . (A) Twenty μg of kidney lysates from vehicle ("-") and insulin("+") -injected 5-6 wk Irs2 +/+ and Irs2 -/- mice were separated on 10% SDS-PAGE and probed by Western blotting with anti-phospho-PKB/Akt Thr308, anti-phospho-PKB/Akt Ser473 and anti-PKB/Akt. GAPDH was used as a loading control. (B, C) Band intensities were quantified using Scion Image software. The intensity ratio of pThr308/GAPDH and pSer473/GAPDH are shown. * p

Article Snippet: Antibodies at the following dilutions were used: IRS-2 (1:500 generated in-house, Prof. Morris White), IRS-2 (Millipore, used for immunoprecipitation), polyclonal pAkt Ser473 (1:1000, Cell Signaling), pAkt Thr 308 (1:1000, Cell Signaling), pGSK-3β Ser9 (1:1000, Cell Signaling), total GSK-3β (1:1000, Cell Signaling), β-catenin (1:1000, BD Biosciences), total YAP (1:1000, Cell Signaling), pYAP Ser127 (1:1000, Cell Signaling), isoform specific PKB/Akt antibodies PKBα (1:500), PKBβ (1:500), PKBγ (1:5000) were a generous gift from Dr. Brian Hemmings, Friedrich Miescher Institute, Basel, Switzerland, total PKB/Akt (1:1000, Cell Signaling), GAPDH (1:5000, Cell Signaling), β-actin (1:25,000, Sigma).

Techniques: Activation Assay, Injection, Mouse Assay, SDS Page, Western Blot, Software

Journal: Journal of Cell Science

Article Title: TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules

doi: 10.1242/jcs.168724

Figure Lengend Snippet: Rictor and Sin1 mutant alleles. (A) Schematic representation of the Rictor locus and the mutant alleles Rictor 77A and Rictor 305A . (B) PCR product of the Rictor open reading frame (ORF) amplified from cDNA of Rictor mutant and control flies. In the deletion mutants, the length of the Rictor ORF is 757 bp shorter than in the control, resulting in a premature stop codon after 58 amino acids. (C) Western blot visualization of Akt phosphorylation (p-Akt) on S505 in lines 1A (control), the Rictor 77A mutant, the Sin1 mutant, the double Rictor 77A ; Sin1 mutant and in the Rictor 77A carrying a rescuing a Rictor transgene ( da > Rictor ). Note that p-Akt is reduced in Rictor and Sin1 single mutants and in Rictor ; Sin1 double mutants. Reduced phosphorylation is rescued with ubiquitous expression of Rictor .

Article Snippet: Phospho-S505 PKB/AKT (1:1000; Cell Signaling, BioConcept, Allschwil, Switzerland), total AKT (Cell Signaling, BioConcept, Allschwil, Switzerland), phospho-p38 (1:1000; Cell Signaling, BioConcept, Allschwil, Switzerland), active JNK (1:3000; Promega, Dübendorf, Switzerland), Erk1/2 (1:500; Sigma, Buchs, Switzerland) and Tubulin (1:10,000; Sigma, Buchs, Switzerland) antibodies were used.

Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules

doi: 10.1242/jcs.168724

Figure Lengend Snippet: Akt phosphorylation on S505 and stability upon heat stress are TORC2 dependent. (A) Western blot of Akt phosphorylated on S505 (p-Akt)and total Akt in lysates of control and Rictor 77A homozygous mutant larvae upon heat exposure for up to 2 h. Note that in Rictor mutants, the heat-induced Akt phosphorylation is not observed and that Akt is lost upon heat stress. (B) Western blot of p-Akt in lysates of S2 cells upon heat stress at 37°C for increasing time (up to 2 h). (C) Western blot of p-Akt in GFP -, Rictor - and Sin1 -depleted S2 cells exposed at 37°C for 2 h. Note that p-Akt does not increase upon loss of TORC2 function. The lower band in this blot is non specific. (D) Western blot of total Akt in GFP -, Rictor - and Sin1 -depleted S2 cells exposed to 26°C and 37°C for 2 h. Note that the Akt level is similar for all conditions at 26°C as well as for mock-depleted conditions at 37°C but dramatically drops in heat-exposed Rictor - and Sin1 -depleted cells.

Article Snippet: Phospho-S505 PKB/AKT (1:1000; Cell Signaling, BioConcept, Allschwil, Switzerland), total AKT (Cell Signaling, BioConcept, Allschwil, Switzerland), phospho-p38 (1:1000; Cell Signaling, BioConcept, Allschwil, Switzerland), active JNK (1:3000; Promega, Dübendorf, Switzerland), Erk1/2 (1:500; Sigma, Buchs, Switzerland) and Tubulin (1:10,000; Sigma, Buchs, Switzerland) antibodies were used.

Techniques: Western Blot, Mutagenesis

Journal: PLoS ONE

Article Title: Identification of the Amino Acids 300-600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

doi: 10.1371/journal.pone.0043296

Figure Lengend Snippet: Inhibition of Akt/PKB reduces IGF-1-induced phosphorylation of serine 573 and 14-3-3 binding. A . Flp-In HEK293 cells stably expressing GFP-IRS2 were starved for serum and incubated for 30 min with 50 ng/ml IGF-1 or 1 µM Akti-1/2 alone, or Akti-1/2 preincubation followed IGF-1 stimulation. 100 µg of total protein was separated on 7.5% SDS gels and membranes were probed with p-Ser-573 and p-Thr-308 of Akt/PKB. Membranes were stripped and reprobed with respective antibodies for detection of protein levels. B . Effect of Akt/PKB inhibition on serine 573 phosphorylation was assessed by scanning densitometry of blots and normalization for protein (mean ± SEM; n = 3; *p

Article Snippet: IRS-2 polyclonal protein antibody (06–506) was from Millipore (Schwalbach, Germany), Akt/PKB protein antibody (610861) from BD Transduction Laboratories (Erembodegem, Belgium), GFP (green fluorescent protein) polyclonal protein antibody (8334), 14-3-3 (C-17) (732) and 14-3-3 K-19 (629) from Santa Cruz (Santa Cruz, USA) and phosphospecific Thr-308-Akt/PKB (9275) as well as Ser-473-Akt/PKB (9271) from Cell Signaling (Boston, USA).

Techniques: Inhibition, Binding Assay, Stable Transfection, Expressing, Incubation

Journal: PLoS ONE

Article Title: Identification of the Amino Acids 300-600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

doi: 10.1371/journal.pone.0043296

Figure Lengend Snippet: Insulin induces binding of 14-3-3 to endogenous IRS-2 and phosphorylates Ser-573 on IRS-2. A. Fao cells were starved for serum overnight and incubated for 30 min with either 10 nM insulin or 50 ng/ml IGF-1. 250 µg protein was immunoprecipitated with IRS-2 antibody and separated on a 5–15% gradient gel. Overlay assay followed stripping and reprobing with IRS-2 antibody as loading control. Successful stimulation is shown as phosphorylation of p-Thr-308 and corresponding Akt/PKB reblot. B. Fao cells were starved for serum overnight and stimulated with 10 nM insulin for the indicated time points. 100 µg of protein was separated on a 7.5% gel and membranes were probed with specific antibodies against p-Ser-573 of IRS-2 and p-Thr-308 of Akt/PKB. For loading control membranes were stripped and reprobed with protein antibody.

Article Snippet: IRS-2 polyclonal protein antibody (06–506) was from Millipore (Schwalbach, Germany), Akt/PKB protein antibody (610861) from BD Transduction Laboratories (Erembodegem, Belgium), GFP (green fluorescent protein) polyclonal protein antibody (8334), 14-3-3 (C-17) (732) and 14-3-3 K-19 (629) from Santa Cruz (Santa Cruz, USA) and phosphospecific Thr-308-Akt/PKB (9275) as well as Ser-473-Akt/PKB (9271) from Cell Signaling (Boston, USA).

Techniques: Binding Assay, Incubation, Immunoprecipitation, Overlay Assay, Stripping Membranes

Journal: PLoS ONE

Article Title: Identification of the Amino Acids 300-600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

doi: 10.1371/journal.pone.0043296

Figure Lengend Snippet: Ser-573 influences phosphorylation of Akt/PKB. A . HEK293 cells transiently expressing GFP-IRS2 or GFP-IRS2-S573A were stimulated with 50 ng/ml IGF-1 for the indicated time points. 40 µg of total protein was separated on 7.5% SDS gels and membranes were incubated with GFP antibody to ensure equal expression levels and with p-Thr-308 and p-Ser-473 antibody respectively. Corresponding Akt/PKB reblots are shown. B . Densitometric analyses of Akt/PKB phosphorylation. Black diamonds represent IRS-2 wild type, white squares IRS2-S573A mutant. Phosphorylation was normalized against total protein and IRS-2 wild type stimulated with IGF-1 for 5 min was set as 1 (mean ± SEM; n = 3; *p

Article Snippet: IRS-2 polyclonal protein antibody (06–506) was from Millipore (Schwalbach, Germany), Akt/PKB protein antibody (610861) from BD Transduction Laboratories (Erembodegem, Belgium), GFP (green fluorescent protein) polyclonal protein antibody (8334), 14-3-3 (C-17) (732) and 14-3-3 K-19 (629) from Santa Cruz (Santa Cruz, USA) and phosphospecific Thr-308-Akt/PKB (9275) as well as Ser-473-Akt/PKB (9271) from Cell Signaling (Boston, USA).

Techniques: Expressing, Incubation, Mutagenesis

Journal:

Article Title: p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK

doi: 10.1073/pnas.0703423105

Figure Lengend Snippet: Activation of Akt/PKB in response to IR requires DNA-PK. ( A ) Embryonal mouse fibroblasts (MEF) from healthy donors and from mice with SCID were irradiated, harvested at the indicated time points, fractionated into nuclei and cytoplasm, and probed for

Article Snippet: The following antibodies were used: Ab-2 (anti-p53; Oncogene Science), 15G8 (anti-phospho-serine-15/p53; Cell Signaling Technology), anti-phospho-serine-20/p53 (Cell Signaling Technology), clone 7 (anti-GSK-3β; BD Transduction Laboratories), 244F9 and 193H12 (anti-phospho-threonine-308/Akt and anti-phospho-serine-473/Akt; Cell Signaling Techmology), anti Akt/PKB and anti-Akt-2/PKBβ (Cell Signaling Technology), M2 (anti-Flag; Sigma), 4B2 (anti-Mdm2; Calbiochem), PC10 (anti-PCNA; Santa Cruz Biotechnology), anti-GST antibody (Rockland), anti-PARP (Roche), G-4 (anti-DNA-PK; Santa Cruz Biotechnology), anti-ATR (N-19; Santa Cruz Biotechnology), anti-phosphatidylinositol 3-kinase p110α (Cell Signaling Technology), and anti-paxillin (BD Biosciences).

Techniques: Activation Assay, Mouse Assay, Irradiation

Journal:

Article Title: p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK

doi: 10.1073/pnas.0703423105

Figure Lengend Snippet: Akt/PKB phosphorylates GSK-3β in response to IR. ( A ) Wild-type lymphoblasts were irradiated, harvested at the indicated times, and separated into cytoplasmic and nuclear fractions. Phosphorylation of Akt/PKB at threonine-308 and serine-473, total

Article Snippet: The following antibodies were used: Ab-2 (anti-p53; Oncogene Science), 15G8 (anti-phospho-serine-15/p53; Cell Signaling Technology), anti-phospho-serine-20/p53 (Cell Signaling Technology), clone 7 (anti-GSK-3β; BD Transduction Laboratories), 244F9 and 193H12 (anti-phospho-threonine-308/Akt and anti-phospho-serine-473/Akt; Cell Signaling Techmology), anti Akt/PKB and anti-Akt-2/PKBβ (Cell Signaling Technology), M2 (anti-Flag; Sigma), 4B2 (anti-Mdm2; Calbiochem), PC10 (anti-PCNA; Santa Cruz Biotechnology), anti-GST antibody (Rockland), anti-PARP (Roche), G-4 (anti-DNA-PK; Santa Cruz Biotechnology), anti-ATR (N-19; Santa Cruz Biotechnology), anti-phosphatidylinositol 3-kinase p110α (Cell Signaling Technology), and anti-paxillin (BD Biosciences).

Techniques: Irradiation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling *

doi: 10.1074/mcp.M800544-MCP200

Figure Lengend Snippet: d 0 / d 4 (± insulin) ratios of 14-3-3 affinity-purified proteins from unstimulated and insulin-stimulated cells. A , comparison of PKB/Akt and AS160 total protein levels and phosphorylation status in lysates of unstimulated and insulin-stimulated HeLa cells used for 14-3-3 phosphoproteomics screens. B , Coomassie-stained SDS gel of proteins isolated by 14-3-3 capture and release from extracts of unstimulated and insulin-stimulated cells (5 times these amounts were run on a parallel gel and used for the MS analyses). C , 14-3-3 Far-Western overlay assay of proteins isolated by 14-3-3 capture and release from extracts of unstimulated and insulin-stimulated cells. D , thumbnail of the d 0 / d 4 (±insulin) rankings for 14-3-3 affinity-purified proteins where off-scale indicates the proteins that were only detected in the preparation from insulin-stimulated cells. Highlighted in pink are proteins already known to be phosphorylated and to bind to 14-3-3s in response to insulin/IGF1 as well as proteins that were discovered to be insulin/IGF1-responsive in this study. In blue are proteins that we already knew do not respond to insulin/IGF1 as well as proteins found not to be insulin/IGF1-responsive in this study (see text). E , examples of d 0 / d 4 ratio quantification for peptides from CCDC6 ( d 0 / d 4 = 6.4 for this peptide, contributing to the overall ratio of 7.1 for three quantified peptides from this protein ( Table I )) and VASP ( d 0 / d 4 = 1.1 for this peptide with an overall ratio of 1.3 for the protein ( supplemental Table 1 )) showing extracted ion chromatograms after analysis in the LTQ-Orbitrap mass spectrometer.

Article Snippet: Anti-phospho-Erk1/2 (Thr(P)202 /Tyr(P)204 ), anti-phospho-Thr308 PKB, and anti-PKB/Akt were from Cell Signaling Technology.

Techniques: Affinity Purification, Staining, SDS-Gel, Isolation, Mass Spectrometry, Western Blot, Overlay Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling *

doi: 10.1074/mcp.M800544-MCP200

Figure Lengend Snippet: Cellular regulation of 14-3-3 binding of IRS2, CCDC6, ZNRF2, SASH1, PRAS40, VASP, and LSR. HA-IRS2, HA-CCDC6, HA-ZNRF2 (wild type and S19A mutant), HA-SASH1 (wild type and S90A mutant), HA-PRAS40, HA-VASP, and HA-LSR (wild type and S493A mutant) were isolated from transfected HEK293 cells that were stimulated as indicated, and anti-HA immunoprecipitates ( IP ) were analyzed by 14-3-3 overlay and Western blotting. Note that at higher exposures and in other experiments it was clear that 14-3-3 binding is not completely abolished with the ZNRF2 S19A mutation. As controls for the efficacy of stimuli and inhibitors, lysates were analyzed with antibodies against phospho-Thr 308 and phospho-Ser 473 of PKB/Akt, total PKB/Akt, phospho-Erk1/2 ( pErk ), and total Erk1/2. PI3 , PI-103; BID , BI-D1870.

Article Snippet: Anti-phospho-Erk1/2 (Thr(P)202 /Tyr(P)204 ), anti-phospho-Thr308 PKB, and anti-PKB/Akt were from Cell Signaling Technology.

Techniques: Binding Assay, Mutagenesis, Isolation, Transfection, Western Blot

Journal: Human Molecular Genetics

Article Title: Endothelial cells from humans and mice with polycystic kidney disease are characterized by polyploidy and chromosome segregation defects through survivin down-regulation

doi: 10.1093/hmg/ddq470

Figure Lengend Snippet: Effect of VEGF on cell ploidy is reversible by PI3K and Akt/PKB inhibitors. ( A ) Cells with and without 10 μ m acetylcholine (ACh) treatment were analyzed with acetylated α-tubulin (green), pericentrin (red) and DAPI (blue) during resting

Article Snippet: All chemicals were purchased from Sigma, except Akt/PKB inhibitor (Calbiochem, Gibbstown, NJ, USA) and Zm447435 (Tocris Bioscience, Ellisville, MO, USA).

Techniques:

Journal: Molecular and Cellular Biochemistry

Article Title: Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase

doi: 10.1007/s11010-017-2996-y

Figure Lengend Snippet: Dose-dependent effects of IGF1 and insulin on signalling, proliferation, and apoptosis in A549 cells. Cells were exposed to IGF1 or insulin as described for Figs. 2 and 4 , and data are shown as in Fig. 8 for Saos-2/B10 cells. Top panel Western blot showing p-Akt/PKB, p-ERK1/2, bottom panel stimulation of DNA synthesis ( n = 7 in triplicate) and inhibition of apoptosis ( n = 2 in triplicate), expressed relative to control ( log scale ). c denotes control, *** p

Article Snippet: Expression levels of Akt/PKB, ERK1/2, IR, IGF1R, IRS1, actin, and LC3A/B were assessed with specific antibodies [Akt/PKB: BD Transduction Laboratories, San Jose, USA; ERK1/2: Cell Signaling, Danvers, USA; insulin Rβ, IGF1Rβ, and IRS1: Santa Cruz, Dallas, USA; actin: clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany; LC3A/B: (DRU4C) Rabbit mAb, Cell Signaling, Danvers, USA].

Techniques: Western Blot, DNA Synthesis, Inhibition

Journal: Molecular and Cellular Biochemistry

Article Title: Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase

doi: 10.1007/s11010-017-2996-y

Figure Lengend Snippet: Time-dependent activation of Akt/PKB and ERK1/2 by IGF1 and insulin in A549 cells. Representative Western blots are shown on top; quantification of signals from two independent experiments (with duplicates) below. Abundance of p-Akt/PKB and p-ERK1/2 is shown relative to control (4 h). Cells were exposed to vehicle ( empty symbols ), to 1 nmol/l IGF1 ( filled squares ) or to 100 nmol/l insulin ( filled triangles ) as outlined in Fig. 1 b, and incubations were stopped after 10, 30, 120, and 240 min (as in Fig. 3 ). M denotes markers

Article Snippet: Expression levels of Akt/PKB, ERK1/2, IR, IGF1R, IRS1, actin, and LC3A/B were assessed with specific antibodies [Akt/PKB: BD Transduction Laboratories, San Jose, USA; ERK1/2: Cell Signaling, Danvers, USA; insulin Rβ, IGF1Rβ, and IRS1: Santa Cruz, Dallas, USA; actin: clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany; LC3A/B: (DRU4C) Rabbit mAb, Cell Signaling, Danvers, USA].

Techniques: Activation Assay, Western Blot

Journal: Molecular and Cellular Biochemistry

Article Title: Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase

doi: 10.1007/s11010-017-2996-y

Figure Lengend Snippet: Effects of IGF1, insulin, glargine, and FCS compared in Saos-2/B10 cells. Cells were exposed to IGF1, insulin, glargine, or FCS as described for Figs. 2 and 4 . Top panel Western blot showing p-Akt/PKB, p-ERK1/2, bottom panel stimulation of DNA synthesis and inhibition of apoptosis, expressed relative to control ( log scale ), n = 7. c denotes control, *** p

Article Snippet: Expression levels of Akt/PKB, ERK1/2, IR, IGF1R, IRS1, actin, and LC3A/B were assessed with specific antibodies [Akt/PKB: BD Transduction Laboratories, San Jose, USA; ERK1/2: Cell Signaling, Danvers, USA; insulin Rβ, IGF1Rβ, and IRS1: Santa Cruz, Dallas, USA; actin: clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany; LC3A/B: (DRU4C) Rabbit mAb, Cell Signaling, Danvers, USA].

Techniques: Western Blot, DNA Synthesis, Inhibition

Journal: Molecular and Cellular Biochemistry

Article Title: Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase

doi: 10.1007/s11010-017-2996-y

Figure Lengend Snippet: Flow diagram of experimental protocols. Cells were grown in FCS-containing media for 3 days and exposed to serum-free, albumin-containing test media as shown. Apoptosis was assessed after 4 h ( a , “common stop”) by an ELISA detecting cytosolic oligonucleosomes. Insulin/IGF1 signalling was analysed by Western blotting using antibodies against the unphosphorylated and phosphorylated forms of IR-IGF1R-IRS1 (not shown), Akt/PKB and ERK1/2, and actin, with “common stop” ( a ) and with “common start” ( b ) protocols as indicated

Article Snippet: Expression levels of Akt/PKB, ERK1/2, IR, IGF1R, IRS1, actin, and LC3A/B were assessed with specific antibodies [Akt/PKB: BD Transduction Laboratories, San Jose, USA; ERK1/2: Cell Signaling, Danvers, USA; insulin Rβ, IGF1Rβ, and IRS1: Santa Cruz, Dallas, USA; actin: clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany; LC3A/B: (DRU4C) Rabbit mAb, Cell Signaling, Danvers, USA].

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular and Cellular Biochemistry

Article Title: Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase

doi: 10.1007/s11010-017-2996-y

Figure Lengend Snippet: Dose-dependent activation of Akt/PKB and ERK1/2 by IGF1 and insulin in Saos-2/B10 cells. Cells were exposed to IGF1 ( a ) and insulin ( b ) for 30 min as described in Fig. 1 b (common start). Representative Western blots are shown on top , below quantification of at least four independent experiments. Abundance of p-Akt/PKB and p-ERK1/2 is expressed relative to control

Article Snippet: Expression levels of Akt/PKB, ERK1/2, IR, IGF1R, IRS1, actin, and LC3A/B were assessed with specific antibodies [Akt/PKB: BD Transduction Laboratories, San Jose, USA; ERK1/2: Cell Signaling, Danvers, USA; insulin Rβ, IGF1Rβ, and IRS1: Santa Cruz, Dallas, USA; actin: clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany; LC3A/B: (DRU4C) Rabbit mAb, Cell Signaling, Danvers, USA].

Techniques: Activation Assay, Western Blot

Journal: Molecular and Cellular Biochemistry

Article Title: Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase

doi: 10.1007/s11010-017-2996-y

Figure Lengend Snippet: Time-dependent activation of Akt/PKB and ERK1/2 by IGF1 and insulin in Saos-2/B10 cells. Representative Western blots are shown on top ; quantification of signals from at least four independent experiments below. Abundance of p-Akt/PKB and p-ERK1/2 is shown relative to control (4 h). Cells were exposed to vehicle ( empty symbols ), to 1 nmol/l IGF1 ( filled squares ) or to 100 nmol/l insulin ( filled triangles ) as outlined in Fig. 1 b, and incubations were stopped after 10, 30, 120, and 240 min

Article Snippet: Expression levels of Akt/PKB, ERK1/2, IR, IGF1R, IRS1, actin, and LC3A/B were assessed with specific antibodies [Akt/PKB: BD Transduction Laboratories, San Jose, USA; ERK1/2: Cell Signaling, Danvers, USA; insulin Rβ, IGF1Rβ, and IRS1: Santa Cruz, Dallas, USA; actin: clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany; LC3A/B: (DRU4C) Rabbit mAb, Cell Signaling, Danvers, USA].

Techniques: Activation Assay, Western Blot

Journal: Journal of Thrombosis and Haemostasis

Article Title: Non-genomic effects of PPAR? ligands: inhibition of GPVI-stimulated platelet activation

doi: 10.1111/j.1538-7836.2009.03732.x

Figure Lengend Snippet: The peroxisome proliferator-activated receptor- γ (PPAR γ ) ligand rosiglitazone modulates glycoprotein VI (GPVI) signaling. Washed platelets were incubated with rosiglitazone or vehicle control for 3 min and then stimulated with collagen (25 μg mL −1 ) for 90 s. Syk (A), LAT (B), PLCγ2 (C) and PKBα/AKT were immunoprecipitated and immunoblotted to detect phosphotyrosine residues. PKBα/AKT phosphorylation (Ser 473 ) was measured using a phosphospecific antibody. Equivalent protein loading was verified by reprobing for Syk (A), LAT (B), PLCγ2 (C) and PKBα/AKT (D). Densitometry analyzes were performed on replicate experiments using blood from four different donors, and data normalized for protein loading levels [mean ± SEM ( n = 4), t- test * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001].

Article Snippet: Anti-Akt/PKBα was purchased from Upstate Biotechnology (Dundee, Scotland).

Techniques: Incubation, Immunoprecipitation