Journal: Physiological Genomics

Article Title: Changes in the phosphoproteome of brown adipose tissue during hibernation in the ground squirrel, Ictidomys tridecemlineatus

doi: 10.1152/physiolgenomics.00038.2017

Figure Lengend Snippet: Increased phosphorylation of ACC Ser79 in hibernating vs. euthermic ground squirrels. BAT extracts prepared in nondenaturing buffer (10 µg of protein in each lane) were subjected to SDS-PAGE in 7.5% (wt/vol) acrylamide gels and immunoblotted with anti-rat phospho Ser79 ACC1 antibody (at a dilution of 1/1,000), which detects phospho Ser80 in squirrel ACC1. A separate gel was run at the same time with identical amounts of extract for immunoblotting with anti-total ACC antibody and anti-PP2A catalytic subunit antibody (each at a dilution of 1/1,000) as a loading control. Top: blots for 4 individuals (euthermic vs. hibernator). At left, the histogram shows band intensities obtained with anti-phospho ACC for euthermic (white bars) vs. hibernating (black bars) animals relative to band intensities obtained with anti-total ACC. At right, the histogram shows band intensities obtained with anti-total ACC for euthermic (white bars) vs. hibernating (black bars) animals relative to band intensities obtained with anti-PP2A catalytic subunit. The data are mean relative band intensities ± SE, n = 4. Statistically significant differences were assessed by an unpaired Student's 2-sided t-test, *Significant difference compared with the corresponding euthermic value, P < 0.05.

Article Snippet: Anti-rat phospho Ser79 ACC1 rabbit polyclonal antibody (#07-303) and anti-total human ACC1 rabbit monoclonal antibody (#04-322) were from Millipore.

Techniques: Phospho-proteomics, SDS Page, Western Blot, Control

Journal: Biogerontology

Article Title: Curcumin induces multiple signaling pathways leading to vascular smooth muscle cell senescence

doi: 10.1007/s10522-019-09825-2

Figure Lengend Snippet: Analysis of proteins involved in cellular senescence and characteristic for selected signaling pathways. a Western blot analysis of AMPK, p-AMPK, ACC, p-ACC, sirtuin 1, p-sirtuin 1, p300, mTOR p-mTOR, p-S6. GAPDH served as a loading control. The representative WB for at least three independent experiments are shown. c—control, b Densitometric analysis of proteins analyzed by WB. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The primary antibodies used were: anti-ATM (1:500), anti-phospho-ATM Ser1981 (1:500), anti-p300 (1:500) (Abcam, Cambridge, UK), anti-GAPDH (1:50000) (Millipore, Darmstadt, Germany); anti-p21WAF1/Cip1 (1:500) (Sigma-Aldrich, St. Louis, USA); anti-p53 (1:500), (Santa Cruz Biotechnology, Santa Cruz, USA); anti-ATR (1:500), anti-phospho-ATR Ser428 (1:500), anti-phospho-p53 Ser15 (1:250), anti-acetyl-p53 Lys382 (1:200), anti-SIRT1 (1:250), anti-phospho-SIRT1 Ser47 (1:250), anti-p38 MAPK (1:500), anti-phospho-p38 MAPK Thr180/Tyr182 (1:500), anti-phospho-MAPKAPK-2 Thr334 (1:500), anti-AMPKα (1:500), anti-phospho-AMPKα Thr172 (1:1000), anti-ACC (1:500), anti-phospho-ACC Ser79 (1:1000), anti-mTOR (1:500), anti-phospho-mTOR Ser2448 (1:500), anti-phospho-S6 Ser235/236 (1:1000) (Cell Signaling Technology, Denvers, USA), anti-Rb (1:250) (NeoMarkers, Fremont, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Stearoyl-CoA Desaturase-1 (SCD1) Augments Saturated Fatty Acid-Induced Lipid Accumulation and Inhibits Apoptosis in Cardiac Myocytes

doi: 10.1371/journal.pone.0033283

Figure Lengend Snippet: (A): FA oxidation was significantly increased in rat neonatal cardiac myocytes treated with 100 µM palmitic acid (PA). (B): On the other hand, glucose oxidation was significantly reduced in cardiac myocytes treated with PA (100 µM). (C): PA (50 to 100 µM) induced excessive FA oxidation was attenuated in cardiac myocytes transduced with a MOI of 20 of Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). (D): PA (50 to 100 µM) -induced suppression of glucose oxidation recovered in cardiac myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). BSA was used as a vehicle. Each sample was counted in a scintillation counter (cpm) and data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA concentration. (E); Phosphorylation levels of AMPK and ACC decreased in cardiac myocytes transduced with an MOI of 20 of Ad-SCD1 in comparison with control cells transduced with Ad-LacZ. Oligomycin (1 µM) was used as an AMPK and ACC activator.

Article Snippet: The mixture was rotated at 4°C for 15 min and centrifuged at 14000 rpm for 15 min. Western blot analysis was performed according to standard procedures using the following primary antibodies: rabbit monoclonal phosphor-AMPK, AMPK, phosphor-ACC, ACC, cleaved-caspase 3, caspase 3 mAb (Cell Signaling) and β-actin (Santa cruz biotechnology, CA, USA).

Techniques: Transduction, Concentration Assay

Journal: Nutrients

Article Title: Sexual Dimorphism in the Selenocysteine Lyase Knockout Mouse

doi: 10.3390/nu10020159

Figure Lengend Snippet: Metabolic characterization of female Scly −/− mice on a Se deficient diet. For metabolic characterization, WT: n = 4, Scly −/− : n = 7. Unless otherwise indicated, mice were 22-weeks-old. ( a ) Body weight of female WT (FWT) and Scly −/− mice at the indicated time points (two-way ANOVA: time F 3,27 = 99.33, p < 0.0001, genotype F 1,27 = 25.38, p = 0.0007, interaction F 3,27 = 13.87, p < 0.0001); ( b ) gonadal WAT (gWAT) expressed as % body weight; ( c ) glucose tolerance test in 20-week-old mice; ( d ) area under the curve (AUC); ( e ) fasting serum insulin. Twenty-two-week old mice were insulin challenged and hepatic expression of ( f ) pAkt (WT: n = 4, Scly −/− : n = 7); ( g ) pAMPK ( n = 4/group); and ( h ) pACC1 ( n = 4/group) was measured. Phosphorylated proteins were normalized to total protein levels. All data are represented as means ± S.E.M (* p < 0.05, *** p < 0.001).

Article Snippet: The antibodies used were anti-pACC1 Ser79, anti-ACC1, anti-pAkt Ser473, anti-Akt, anti-AMPKα, anti-pAMPKα Thr172 (Cell Signaling, Beverly, MA, USA), anti-GPX1 (R&D Systems, Inc., Minneapolis, MN, USA), anti-SELENOM (Sigma-Aldrich, St. Louis, MO, USA), anti-SELENOS (Sigma-Aldrich, St. Louis, MO, USA), anti-SEPHS2 (Rockland, Gilbertsville, PA, USA), and anti-TXNRD1 (NovusBio, Littleton, CO, USA).

Techniques: Expressing