Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. Whole cell lysates from gastrocnemius muscle of either FynKO mice, WT, HSA-FynB (FynB) or HSA-FynT (FynT) mice were homogenized and immnoblots were performed by AMPKα mAb and GAPDH B. Gastrocnemius muscle of either FynKO mice, WT, HSA-FynB (FynB) or HSA-FynT (FynT) mice were homogenized and immunoprecipitated with 4G10 mAb followed by immunoblotting with AMPK mAb. Blots are representative of 3 independent experiments. C. Signal quantification of tyrosine-phosphorylated AMPKα levels from (A) and (B). Statistical examination between each set was performed. *** P < 0.005.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A., B. NIH3T3 cells were co-transfected with 3myc-AMPKα2-Flag and Fyn-CA-V5. 48 h later, cells extracts were prepared and immunoprecipitated with a Flag antibody. Immunoblots of lysates (A) and immunoprecipitation samples (B) were performed with the indicated antibodies. Blots are representative of 3 independent experiments. C., D. Forty-eight hours after co-transfection with 3myc-AMPKα2-Flag, Fyn-CA-V5 and pcDNA3-LKB1, HEK293T cells extracts were prepared and immunoprecipitation was performed using a Flag antibody. Immunoblots of lysates (C) and immunoprecipitation samples (D) were performed with the indicated antibodies. These are representative immunoblots independently performed 3 times. E. Signal quantification of tyrosine-phosphorylated AMPKα levels from (D). Statistical examination between each set was performed. F. Purified Human PRKAA2 (100 ng) and either mock or Flag-Fyn-CA (380 ng) were incubated with the kinase buffer and ATP at 30°C for 30 min. Samples were boiled and separated onto a 8% SDS-PAGE gel, followed by immunoblotting with the indicated antibodies. G., H. HeLa cells were co-transfected by either 3myc-AMPKα2 WT or Y436F (YF) with Fyn-CA-V5. Immunoprecipitation was performed using a Flag antibody. Immunoblots of lysates (G) and immunoprecipitation samples (H) were performed with the indicated antibodies. Images are representative of 4 independent experiments.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Immunoprecipitation, Western Blot, Cotransfection, Purification, Incubation, SDS Page

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were co-transfected with human siAMPK α1 and α2 subunits and either empty vector (pcDNA3), GST-rat AMPKα WT (WT) or Y430F mutant (equivalent to human Y436F (Y436F)) that are not affected by human AMPKa siRNA. 72 h after transfection immunoblots were performed with the indicated antibodies. B., C. S79 phosphorylation of ACC and total ACC levels in samples from (A) were assessed (B) and signal quantification of the expression levels of S79 phosphorylation of ACC normalized with total ACC were performed (C). Statistical examination between each sets were performed * p < 0.05, *** p < 0.005. D. , E . HEK293T cells were transfected by either 3myc-AMPKα2 WT or Y436F-Flag (Y436F). Co-immunoprecipitation was performed by either control IgG or AMPK β1/β2 polyclonal antibody. Immunoblots of lysates (D) and immunoprecipitation samples (E) were performed with the indicated antibodies. Images are representative of 3 independent experiments.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation, Control

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were incubated with TNFα (10 ng/ml) for 12, 24 or 36 h and then stimulated with 2 mM AICAR for 10 min. Immunoblots were performed with the indicated antibodies. These are representative images from 3 independent experiments. B., C. Signal quantifications of expression levels of phospho- T172 AMPKα corrected by total AMPKα (B) and levels of phospho- S79 of ACC to total ACC (C). The data are presented as mean ± s.e.m. Statistical examination between control and AICAR stimulated cells in each sets were performed. ** p < 0.01, *** p < 0.005.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Incubation, Western Blot, Expressing, Control

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. Fyn kinase activity in HEK293T cells treated or not with TNFα (10ng/ml) for 36 h. These are representative images from 5 independent experiments *** p < 0.005. B. Fyn expression in HEK293T cells treated or not with TNFα for 36 h. C. After a 36-h incubation with 10ng/ml TNFα, HEK293T cells were treated or not with TNFα for 36 h and homogenized. Immunoprecipitation was performed with total AMPKα antibody followed by immunoblotting with the 4G10 and total AMPKα antibody. These are representative images from 4 independent experiments. D. Signal quantification (Y436 normalized to total AMPKα) from (C). (*** P < 0.005).

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Activity Assay, Expressing, Incubation, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were transfected with Fyn siRNAs. Cells were treated with TNFα (10 ng/ml) for 36 h with or without 2 mM AICAR for 10 min. Immunoblots were performed with the indicated antibodies. These are representative images from 3 independent experiments. B., C. Signal quantifications of expression levels of phospho- T172 AMPKα normalized to total AMPKα (B) and levels of S79 phosphorylation of ACC normalized to total ACC (C). The data are presented as mean ± SE. Statistical examination between control and AICAR stimulated cells in each sets were performed. * P < 0.05, ** P < 0.01.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were co-transfected with siRNAs of human siAMPK α1 and α2 subunits and either empty vector (pcDNA3), GST-rat AMPKα WT (WT) or Y430F mutant (equivalent to human Y436F (Y436F)) to be replaced. Immunoblots were performed 72 h after transfection. B. HEK293T cells were transfected with Fyn siRNAs. Cells were treated with 10ng/ml TNFα for 36 h with or without 2 mM AICAR for 10 min. Immunoblots were performed with the indicated antibodies. These are representative images from 3 independent experiments. C. Signal quantifications of the expression levels of phospho-S555 ULK1 to total ULK1. The data are presented as mean ± SE. Statistical examination between control and AICAR stimulated cells in each sets were performed * P < 0.05, ** P < 0.01.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, Control

Journal: eLife

Article Title: Obesity-linked suppression of membrane-bound O -acyltransferase 7 (MBOAT7) drives non-alcoholic fatty liver disease

doi: 10.7554/eLife.49882

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-GPAT4 (Rabbit Polyclonal) , Novus BIologicals , Cat#: NB100-2390; RRID: AB_2273811 , 1:1000.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Recombinant