Journal: Biochimica et biophysica acta

Article Title: 5'-AMP-activated protein kinase alpha regulates stress granule biogenesis.

doi: 10.1016/j.bbamcr.2015.03.015

Figure Lengend Snippet: Fig. 5. Compound C impairs SG formation. (A) HeLa cells were pre-incubated with the vehicle DMSO or CC for 1 h; DMSO or CC was also present throughout the subsequent treatment period. SG assembly was induced by arsenite; the vehicle water was added to controls. Alternatively, SGs were produced with DEM, whereas controls received ethanol. Both AMPK-α iso- forms (AMPK-α1/2) were detected in SGs demarcated with the marker HuR. (B) The same results as in part A were obtained with the SG marker G3BP1. Scale bar is 20 μm. (C) Western blots for control and DEM-treated samples show a reduction of α-subunit phosphorylation (α1, Thr183; α2, Thr172) after DEM incubation. CC further diminishes this modification. The loss of AMPK kinase activity was verified by probing for phosphorylated Acc1 (p-Acc1). Phosphorylation of eIF2α on Ser51 was not abolished by DEM or CC. DEM or CC had also no strong effects on the cellular concentrations of TIA-1/TIAR, G3BP1 or HuR.

Article Snippet: Primary antibodies were used at the following dilutions: AMPK-α1/2 (1:200), p-AMPK-α (1:2000; Cell Signaling #2535), p-Acc1 (1:1000; phosphoSer79; Cell Signaling #3661); Acc1 (1:500; Cell Signaling #3662), HuR (1:2000), G3BP1 (1:2000), TIA-1/TIAR (1:1000), AMPKα1 and AMPK-α2 (1:2000), and actin (1:100,000).

Techniques: Incubation, Produced, Marker, Western Blot, Control, Phospho-proteomics, Activity Assay

Journal: Biochimica et biophysica acta

Article Title: 5'-AMP-activated protein kinase alpha regulates stress granule biogenesis.

doi: 10.1016/j.bbamcr.2015.03.015

Figure Lengend Snippet: Fig. 11. AMPK activation precedes SG formation. HeLa cells were incubated with DEM for the time points indicated. (A) AMPK-α phosphorylation (p-AMPK-α1/2), total AMPK-α1/2, phosphorylated Acc1 (p-Acc1, Ser79) and total Acc1 were detected by Western blotting. Controls were incubated with the vehicle ethanol (EtOH). (B) The kinetics for SG formation was monitored by immunodetection of AMPK-α2 and G3BP1. SGs were formed after 60 min incubation with DEM. They contained the marker G3BP1 and AMPK-α2.

Article Snippet: Primary antibodies were used at the following dilutions: AMPK-α1/2 (1:200), p-AMPK-α (1:2000; Cell Signaling #2535), p-Acc1 (1:1000; phosphoSer79; Cell Signaling #3661); Acc1 (1:500; Cell Signaling #3662), HuR (1:2000), G3BP1 (1:2000), TIA-1/TIAR (1:1000), AMPKα1 and AMPK-α2 (1:2000), and actin (1:100,000).

Techniques: Activation Assay, Incubation, Phospho-proteomics, Western Blot, Immunodetection, Marker

Journal: Antioxidants

Article Title: Cranberry Proanthocyanidins as a Therapeutic Strategy to Curb Metabolic Syndrome and Fatty Liver-Associated Disorders

doi: 10.3390/antiox12010090

Figure Lengend Snippet: PAC modulates hepatic lipid metabolism. Protein expression of pivotal biomarkers influencing lipid metabolism and biogenesis was determined in liver by Western blot as described in Materials and Methods. ( A ) ACC, ( B ) p-ACC, ( D ) FAS, ( E ) SREBP1c, ( F ) CPT1A, ( G ) PPARα, ( H ) PGC1α, ( I ) AMPKα and ( J ) p-AMPKα protein mass was determined. ( A ) ACC and ( B ) p-ACC as well as ( I ) AMPKα and ( J ) p-AMPKα were blotted on separate gels and normalized using their respective β-actins; the ( C ) p-ACC/ACC and ( K ) p-AMPKα/AMPKα ratios were then calculated. Data are expressed as the mean ± SEM for n = 4–8 mice/group with a representative gel per experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFHS mice. Each set of experiments, including the proteins of interest and their β-actin (as a reference protein and a loading housekeeping control) was run on the same gel. ACC: acetyl-CoA carboxylase; p-ACC: Phosphorylated-acetyl-CoA carboxylase; FAS: Fatty acid synthase; SREBP1c: Sterol regulatory element binding protein-1c; CPT1A: Carnitine palmitoyl transferase 1 isoform A; PPARα: Peroxisome proliferator activated receptor alpha; PGC1α: Peroxisome proliferator activated receptor gamma coactivator 1 alpha; AMPKα: AMP-activated protein kinaseα; p-AMPKα: Phosphorylated-AMP-activated protein kinaseα.

Article Snippet: Fat-free milk was used for the initial blocking of non-specific sites, followed by overnight incubation at 4 °C with the following primary antibodies at 1/1000 dilution unless otherwise specified: Sterol regulatory element binding protein-1c (SREBP1c), Peroxisome proliferator-activated receptor alpha (PPARα) from Cayman Chemical); Fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), AMP-activated protein kinaseα (AMPKα) and its phosphorylated form Phosphorylated AMPKα Thr172 (p-AMPKα), Carnitine palmitoyl transferase 1 isoform A (CPT1A), Inhibitor of kappa B (IκB; 1/500) from Cell signalling; Peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α), Nuclear factor erythroid-2-related factor 2 (NRF2) from Abcam; Superoxide dismutase 2 (SOD2), Carbohydrate response element binding protein (ChREBP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Invitrogen; Glutathione peroxidase 1 (GPx), Cyclooxygenase-2 (COX-2) from Novus Biologicals; Nuclear factor kappa B (NF-κB; 1/250), Glucose 6-phosphatase (G6Pase), Phosphoenolpyruvate carboxykinase (PEPCK) from Santa Cruz Biotechnology; Phosphorylated acetyl-CoA carboxylase Ser79 (p-ACC; 1/500, Millipore); Tumor necrosis factor-alpha (TNFα; ThermoFisher Scientific, Waltham, MA, USA) and β-actin (1/250,000; Sigma-Aldrich, Burlington, MA, USA).

Techniques: Expressing, Western Blot, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Effect of His Bundle Pacing on Abnormal Myocardial Fatty Acid and Glucose Metabolism Induced by Right Ventricular Pacing

doi: 10.1161/JAHA.123.032386

Figure Lengend Snippet: In terms of regulatory proteins related to fatty acid metabolism, we checked the key proteins, such as CD36, CPT1, ACC1 and HSL. A , The expression of CD36 was higher in the RV pacing group than the sham control and His pacing groups. The expression of CPT1 ( B ) and p‐ACC1/ACC1 ( C ) did not differ among the 3 groups. D , The expression of HSL was significantly lower in the RV pacing group compared with the sham control and His pacing groups. E , We also evaluated the inhibitory form of HSL ( p565 HSL), and the expression of p565 HSL/HSL was significantly lower in the RV pacing group compared with the sham control and His pacing groups. * P <0.05; ** P <0.01. ACC1 indicates acetyl‐CoA carboxylase 1; CPT1, carnitine palmitoyltransferase I; HSL, hormone‐sensitive lipase; and p‐ACC1, phosphorylated acetyl‐CoA carboxylase 1.

Article Snippet: The primary antibodies against binding immunoglobulin protein (Bip), glucose transporter 4 (GLUT4), hormone‐sensitive lipase (HSL), pyruvate dehydrogenase (PDH), acetyl‐CoA carboxylase 1 (ACC1), phosphorylated ACC1 (Ser79), phosphorylated HSL (Ser565) (1:1000 dilution; Cell Signaling Technology, Danvers, MA), tumor necrosis factor‐α (TNFα; 1:200 dilution; Santa Cruz, TX), and carnitine palmitoyltransferase I (CPT1) (1:5000 dilution; Abcam, Cambridge, MA) were used to react with the blots at 4 °C overnight in 5% nonfat dry milk.

Techniques: Expressing