ampk signaling phosphorylation antibody array  (Full Moon BioSystems)

Journal: Journal of Advanced Research

Article Title: Metrnl ameliorates diabetic cardiomyopathy via inactivation of cGAS/STING signaling dependent on LKB1/AMPK/ULK1-mediated autophagy

doi: 10.1016/j.jare.2022.10.014

Figure Lengend Snippet: Metrnl activates the LKB1/AMPK signaling in cardiomyocytes . ( A ) Heatmap showing the effects of Metrnl on the components of AMPK array. ( B ) Top ten signaling protein induced by Metrnl. ( C ) Metrnl dose- and time-dependently induced AMPK phosphorylation. ( D ) Effects of Metrnl on HG-induced downregulation of phosphorylated AMPK. ( E ) Effects of Metrnl shRNA on HG-induced downregulation of phosphorylated AMPK. ( F ) Cardiomyocytes were pretreated with STO-609 (0.8 μM) or Takinib (10 mM) for 30 min, and challenged by Metrnl (0.2 μg/ml) for 30 min, and then stimulated for HG for 48 h. Cardiomyocytes were pretreated with LKB1 siRNA (100 nM) for 6 h, and challenged by Metrnl (0.2 μg/ml) for 30 min, and then stimulated for HG for 48 h. Western blot was used to determine phosphorylated AMPK. ( G ) Effects of Metrnl on HG-induced downregulation of phosphorylated LKB1. ( H ) Effects of Metrnl shRNA on HG-induced downregulation of phosphorylated LKB1. n = 4. * P < 0.05 versus 0 μg/ml, 0 min, NG, Con, Con shRNA, † P < 0.05 versus HG + Con shRNA, Metrnl or HG.

Article Snippet: The cell lysates from four-independent cell samples in each group were collected for the antibody array experiments using an AMPK signaling phosphorylation antibody array (PAM174, Full Moon Biosystems, Sunnyvale, USA).

Techniques: shRNA, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis *

doi: 10.1074/jbc.M112.348946

Figure Lengend Snippet: CCL5 induces phosphorylation of proteins in the AMPK signaling pathway. A, the Full Moon BioSystems AMPK signaling phospho-specific antibody array includes six replicates (vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway. Biotinylated protein lysates were added to microscope slide chambers and fluorescence from Cy3-streptavidin was measured with the Axon GenePix 400A microarray scanner. B, the extent of protein phosphorylation (mean fluorescence intensity, MFI) was normalized within each slide and compared between untreated control and cells treated with 10 nm CCL5 for 10 min. The data are represented as fold CCL5-induction relative to untreated controls. Phosphorylated signaling intermediates associated with metabolism are indicated (red arrows).

Article Snippet: A , the Full Moon BioSystems AMPK signaling phospho-specific antibody array includes six replicates (vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway.

Techniques: Phospho-proteomics, Ab Array, Microscopy, Fluorescence, Microarray, Control

Journal: The Journal of Biological Chemistry

Article Title: The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis *

doi: 10.1074/jbc.M112.348946

Figure Lengend Snippet: CCL5 activates the energy-sensing kinase AMPK and the downstream substrate GSK-3β resulting in an increased intracellular ATP levels. A, activated PB T cells were either left untreated or treated with 10 nm CCL5 for the indicated times. Cells were harvested and protein lysates resolved by SDS-PAGE and immunoblotted with anti-phospho-AMPKα (Thr-172) or anti-phospho-GSK-3β (Ser-9) antibodies. Membranes were stripped and reprobed for loading. Relative phosphorylation is shown as signal intensity over loading control. Data are representative of two independent experiments. B, activated PB T cells were either treated with dimethyl sulfoxide or 10 μm compound C for 1 h before treatment with 10 nm CCL5 for the indicated times. Intracellular ATP was measured using a bioluminescent assay. Data are representative of two independent experiments. *, p < 0.01; **, p < 0.05. C, activated PB T-cells were pretreated with dimethyl sulfoxide (DMSO; carrier control), oligomycin (1 μm), or 2-deoxy-glucose (10 mm) for 30 min and then stimulated with IL-2 (20 ng/ml) or CCL5 (10 nm) for 30 min. Intracellular ATP levels were then measured using a bioluminescent assay. Data are representative of two independent experiments. *, p < 0.01.

Article Snippet: A , the Full Moon BioSystems AMPK signaling phospho-specific antibody array includes six replicates (vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway.

Techniques: SDS Page, Phospho-proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis *

doi: 10.1074/jbc.M112.348946

Figure Lengend Snippet: Glucose uptake and AMPK signaling are required for efficient CCL5-mediated chemotaxis. A, activated PB T cells were either left untreated or pretreated with 2-DG at the doses indicated for 1 h. A total of 1 × 105 cells in 100 μl of chemotaxis buffer were then placed in the upper chamber of Transwell chambers. CCL5-mediated chemotaxis was measured using 10 nm CCL5. Data are presented as % migration, with the number of migrated cells at 10 nm CCL5 taken as 100%. Data are representative of three independent experiments. B, activated PB T cells were pre-treated with either dimethyl sulfoxide (DMSO; carrier) or different doses of compound C for 1 h. CCL5-mediated chemotaxis was measured as described in A. Data are representative of three independent experiments. *, p < 0.01.

Article Snippet: A , the Full Moon BioSystems AMPK signaling phospho-specific antibody array includes six replicates (vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway.

Techniques: Chemotaxis Assay, Migration

Journal: The Journal of Biological Chemistry

Article Title: The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis *

doi: 10.1074/jbc.M112.348946

Figure Lengend Snippet: CCL5 induces phosphorylation of proteins in the AMPK signaling pathway. A, the Full Moon BioSystems AMPK signaling phospho-specific antibody array includes six replicates (vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway. Biotinylated protein lysates were added to microscope slide chambers and fluorescence from Cy3-streptavidin was measured with the Axon GenePix 400A microarray scanner. B, the extent of protein phosphorylation (mean fluorescence intensity, MFI) was normalized within each slide and compared between untreated control and cells treated with 10 nm CCL5 for 10 min. The data are represented as fold CCL5-induction relative to untreated controls. Phosphorylated signaling intermediates associated with metabolism are indicated (red arrows).

Article Snippet: AMPK Signaling Antibody Array Phosphorylation events in the AMPK signaling pathway were examined using the Full Moon BioSystems Antibody Microarray, according to the manufacturer's specifications (Full Moon BioSystems, Inc.) Briefly, 5 × 10 6 cells were stimulated with CCL5 for 10 min, washed with ice-cold PBS, and lysed with 200 μl of extraction buffer.

Techniques: Phospho-proteomics, Ab Array, Microscopy, Fluorescence, Microarray, Control

Journal: The Journal of Biological Chemistry

Article Title: The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis *

doi: 10.1074/jbc.M112.348946

Figure Lengend Snippet: CCL5 activates the energy-sensing kinase AMPK and the downstream substrate GSK-3β resulting in an increased intracellular ATP levels. A, activated PB T cells were either left untreated or treated with 10 nm CCL5 for the indicated times. Cells were harvested and protein lysates resolved by SDS-PAGE and immunoblotted with anti-phospho-AMPKα (Thr-172) or anti-phospho-GSK-3β (Ser-9) antibodies. Membranes were stripped and reprobed for loading. Relative phosphorylation is shown as signal intensity over loading control. Data are representative of two independent experiments. B, activated PB T cells were either treated with dimethyl sulfoxide or 10 μm compound C for 1 h before treatment with 10 nm CCL5 for the indicated times. Intracellular ATP was measured using a bioluminescent assay. Data are representative of two independent experiments. *, p < 0.01; **, p < 0.05. C, activated PB T-cells were pretreated with dimethyl sulfoxide (DMSO; carrier control), oligomycin (1 μm), or 2-deoxy-glucose (10 mm) for 30 min and then stimulated with IL-2 (20 ng/ml) or CCL5 (10 nm) for 30 min. Intracellular ATP levels were then measured using a bioluminescent assay. Data are representative of two independent experiments. *, p < 0.01.

Article Snippet: AMPK Signaling Antibody Array Phosphorylation events in the AMPK signaling pathway were examined using the Full Moon BioSystems Antibody Microarray, according to the manufacturer's specifications (Full Moon BioSystems, Inc.) Briefly, 5 × 10 6 cells were stimulated with CCL5 for 10 min, washed with ice-cold PBS, and lysed with 200 μl of extraction buffer.

Techniques: SDS Page, Phospho-proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis *

doi: 10.1074/jbc.M112.348946

Figure Lengend Snippet: Glucose uptake and AMPK signaling are required for efficient CCL5-mediated chemotaxis. A, activated PB T cells were either left untreated or pretreated with 2-DG at the doses indicated for 1 h. A total of 1 × 105 cells in 100 μl of chemotaxis buffer were then placed in the upper chamber of Transwell chambers. CCL5-mediated chemotaxis was measured using 10 nm CCL5. Data are presented as % migration, with the number of migrated cells at 10 nm CCL5 taken as 100%. Data are representative of three independent experiments. B, activated PB T cells were pre-treated with either dimethyl sulfoxide (DMSO; carrier) or different doses of compound C for 1 h. CCL5-mediated chemotaxis was measured as described in A. Data are representative of three independent experiments. *, p < 0.01.

Article Snippet: AMPK Signaling Antibody Array Phosphorylation events in the AMPK signaling pathway were examined using the Full Moon BioSystems Antibody Microarray, according to the manufacturer's specifications (Full Moon BioSystems, Inc.) Briefly, 5 × 10 6 cells were stimulated with CCL5 for 10 min, washed with ice-cold PBS, and lysed with 200 μl of extraction buffer.

Techniques: Chemotaxis Assay, Migration