Journal: The Journal of Biological Chemistry

Article Title: Akt Cys-310-targeted Inhibition by Hydroxylated Benzene Derivatives Is Tightly Linked to Their Immunosuppressive Effects *

doi: 10.1074/jbc.M109.074872

Figure Lengend Snippet: The effect of HQ on NF-κB translocation signaling activation. RAW264.7 cells (A, B, left panel, and C) or peritoneal macrophages (B, right panel) (5 × 106 cells/ml) were incubated with HQ in the presence or absence of LPS (2 μg/ml) for 30 min. The levels of phosphorylated or total p85, PDK1, Akt, MEK, and MAPK (ERK, JNK, and p38) from total lysates were analyzed by immunoblotting. The results show one representative experiment of three.

Article Snippet: Phospho- or total antibodies to p85, 3-phosphoinositide-dependent kinase 1 (PDK1), Akt (protein kinase B) (Thr-308 and Ser-473), extracellular signal-regulated kinase (ERK), ERK kinase (MEK), p38, c-Jun N-terminal kinase (JNK), IκBα kinase (IKK), IκBα, p65, myelin basic protein (MBP), γ-tubulin, and β-actin were purchased from Cell Signaling (Beverly, MA), Santa Cruz Biotechnology (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Translocation Assay, Activation Assay, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Akt Cys-310-targeted Inhibition by Hydroxylated Benzene Derivatives Is Tightly Linked to Their Immunosuppressive Effects *

doi: 10.1074/jbc.M109.074872

Figure Lengend Snippet: The effect of HQ on kinase activity of Akt and PDK1. A, shown are amino acid sequences of Suntide and Crosstide. B, HQ was incubated with purified Akt prepared from the baculovirus system for 20 min. Kinase activity was measured with Crosstide (30 μm) as described under “Experimental Procedures.” C, 2A1 cells (5 × 106 cells/ml) pretreated with HQ for 15 or 30 min were incubated in the presence or absence of insulin (100 μm) for 20 min. After immunoblotting, total or phospho-protein levels of Akt were identified with the total protein- or phospho-specific antibodies. D, HQ was incubated with immunoprecipitated PDK1 prepared from Myc-PDK1 plasmid (1 μg/ml)-transfected HEK293 cells (5 × 106 cells/ml) for 20 min. Kinase activity was measured with Suntide (100 μm) or MBP (10 ng/ml) as described under “Experimental Procedures.” E, HQ was incubated with immunoprecipitated PDK1 prepared from LPS-activated RAW264.7 cells (5 × 106 cells/ml) for 10 or 15 min. Kinase activity was measured with MBP (10 ng/ml) as described under “Experimental Procedures.” Data (B) represent the mean ± S.E. of three independent experiments performed in triplicate. The results (C–E) show one representative duplicate experiment of three. p < 0.05 (*) and p < 0.01 (**) compared with the control group.

Article Snippet: Phospho- or total antibodies to p85, 3-phosphoinositide-dependent kinase 1 (PDK1), Akt (protein kinase B) (Thr-308 and Ser-473), extracellular signal-regulated kinase (ERK), ERK kinase (MEK), p38, c-Jun N-terminal kinase (JNK), IκBα kinase (IKK), IκBα, p65, myelin basic protein (MBP), γ-tubulin, and β-actin were purchased from Cell Signaling (Beverly, MA), Santa Cruz Biotechnology (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activity Assay, Incubation, Purification, Western Blot, Immunoprecipitation, Plasmid Preparation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Akt Cys-310-targeted Inhibition by Hydroxylated Benzene Derivatives Is Tightly Linked to Their Immunosuppressive Effects *

doi: 10.1074/jbc.M109.074872

Figure Lengend Snippet: The effect of thiol compounds on HQ-mediated inhibition of Akt phosphorylation and kinase activity. A, RAW264.7 cells (5 × 106 cells/ml) pretreated with l-cysteine were incubated with HQ in the presence or absence of LPS (2 μg/ml) for 5 and 30 min. The phosphorylated or total levels of Akt from total lysates were analyzed by immunoblotting. B, 2A1 cells (5 × 106 cells/ml) pretreated with DTT were stimulated with HQ in the presence or absence of insulin (100 μm). After immunoblotting, total or phosphoprotein levels of Akt were identified with the total protein- or phospho-specific antibodies. C, kinase activity of immunoprecipitated Akt prepared from 2A1 cells (5 × 106 cells/ml) stimulated with insulin (100 μm) was measured with Crosstide (30 μm) after incubation with DTT and HQ for 10 min. D, HQ and DTT were directly incubated with immunoprecipitated PDK1 for 10 min. Kinase activity was measured with Suntide (100 μm). Data (C and D) represent the mean ± S.E. of three independent experiments performed in triplicate. The results (A and B) show one representative experiment of three. p < 0.05 (*) and p < 0.01 (**) compared with the control group. p < 0.05 (#) and p < 0.01 (##) compared with the stimulus + HQ group with the same concentration.

Article Snippet: Phospho- or total antibodies to p85, 3-phosphoinositide-dependent kinase 1 (PDK1), Akt (protein kinase B) (Thr-308 and Ser-473), extracellular signal-regulated kinase (ERK), ERK kinase (MEK), p38, c-Jun N-terminal kinase (JNK), IκBα kinase (IKK), IκBα, p65, myelin basic protein (MBP), γ-tubulin, and β-actin were purchased from Cell Signaling (Beverly, MA), Santa Cruz Biotechnology (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Inhibition, Activity Assay, Incubation, Western Blot, Immunoprecipitation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Akt Cys-310-targeted Inhibition by Hydroxylated Benzene Derivatives Is Tightly Linked to Their Immunosuppressive Effects *

doi: 10.1074/jbc.M109.074872

Figure Lengend Snippet: The role of Cys-310 in the kinase activity of Akt and HQ inhibition. A, amino acid sequences of Akt-WT and the Akt-C310A mutant are shown. PH, pleckstrin homology. PKB, protein kinase B. B, HEK293 cells were transfected with plasmid constructs containing GFP-Akt-WT or GFP-Akt-C310A (1 μg/ml each) for 48 h, and total lysates were prepared after treatment of pervanadate (100 μm) for 15 min. After immunoprecipitation (IP) with an anti-GFP antibody, the phosphorylated or total levels of Akt and GFP were analyzed by immunoblotting (WB). C, HQ was directly incubated with immunoprecipitated Akt-WT or Akt-C310A prepared from HEK293 cells (5 × 106 cells/ml) transfected with GFP-Akt or GFP-Akt-C310A (1 μg/ml) for 10 min. Kinase activity was measured with Crosstide (30 μm). D, a putative docking model between MHQ and Akt Cys-310 is shown. The results (B and C) show one representative duplicate experiment of three. p < 0.05 (*) and p < 0.01 (**) compared with the control group.

Article Snippet: Phospho- or total antibodies to p85, 3-phosphoinositide-dependent kinase 1 (PDK1), Akt (protein kinase B) (Thr-308 and Ser-473), extracellular signal-regulated kinase (ERK), ERK kinase (MEK), p38, c-Jun N-terminal kinase (JNK), IκBα kinase (IKK), IκBα, p65, myelin basic protein (MBP), γ-tubulin, and β-actin were purchased from Cell Signaling (Beverly, MA), Santa Cruz Biotechnology (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Lake Placid, NY).

Techniques: Activity Assay, Inhibition, Mutagenesis, Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Western Blot, Incubation

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Postexercise muscle glycogen synthesis with glucose, galactose, and combined galactose-glucose ingestion

doi: 10.1152/ajpendo.00127.2022

Figure Lengend Snippet: Antibodies used in Western blot analysis

Article Snippet: Protein kinase B phosphorylation site Thr308 , Cell Signaling Technology , 2965 , 1:5,000.

Techniques: Western Blot, Concentration Assay, Phospho-proteomics