active phosphorylated form of amp-activated protein kinase alpha (p-ampk)  (Cell Signaling Technology Inc)

Journal: Antioxidants

Article Title: OR2AT4, an Ectopic Olfactory Receptor, Suppresses Oxidative Stress-Induced Senescence in Human Keratinocytes

doi: 10.3390/antiox11112180

Figure Lengend Snippet: OR2AT4 activation by sandalore mediates the CAMKKβ/AMPK/mTORC1/autophagy axis. Expression of total and phosphorylated ( A ) CAMKKβ and ( B ) AMPK by immunoblot analysis. ( C ) Representative images of MDC-stained HaCaT cells. Scale bar, 20 µm (left panel). Quantification of the MDC fluorescence intensity in HaCaT cells (right panel). Fluorescence was quantified using the VICTOR ™ X Multilabel Plate Reader. ( D ) The expression of LC3I and LC3II by immunoblot analysis (left panel). Bar graphs represent the ratio of the LC3II to LC3I within each sample (right panel). ( E ) Expression of the total and phosphorylated mTORC1 and 70S6K by immunoblot analysis. ( F ) Expression of the total and phosphorylated AMPK in the OR2AT4 -knockdown HaCaT cells. The relative protein expression of p-CAMKKβ/CAMMKβ, p-AMPK/AMPK, p-mTORC1/TORC1, and p-p70S6K/p70S6K were analyzed using ImageLab software. Data are means ± SEM. * p < 0.05, ** p <0.01, and *** p < 0.001. Student’s t -test was performed for comparisons between groups.

Article Snippet: The primary antibodies used for immunoblotting were as follows: OR2AT4 (ab105828, 36 kDa; Abcam, Cambridge, MA, USA), GNAL (sc-48345, 46 kDa; Santa Cruz Biotechnology, Santa Cruz, CA, USA), adenylate cyclase 3 (ADCY3) (sc-588, 130 kDa; Santa Cruz Biotechnology), p21/CIP1/CDKN1A (NBP2-294563, 21 kDa; Novus, St. Louis, MO, USA), phosphorylated calcium/calmodulin-dependent protein kinase kinase (p-CaMKKβ) (12818S, 50 kDa; Cell Signaling Technology, Danvers, MA, USA), CaMKKβ (sc-50341, 50 kDa; Santa Cruz Biotechnology), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) (44-1150G, 62 kDa; Invitrogen, Carlsbad, CA, USA), AMPK (sc-25792, 62 kDa; Santa Cruz Biotechnology), LC3 antibody (sc-0376404, 15 kDa; Santa Cruz Biotechnology), phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1) antibody (ab63552, 289 kDa; Abcam), mTORC1 antibody (2983S, 289 kDa; Cell Signaling Technology), phosphorylated p70 ribosomal S6 kinase (p-p70S6K) antibody (2211S, 80 kDa; Cell Signaling Technology), p70S6K antibody (2217S, 80 kDa; Cell Signaling Technology), and β-actin (sc-47778, 43 kDa; Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Western Blot, Staining, Fluorescence, Knockdown, Software

Journal: Antioxidants

Article Title: OR2AT4, an Ectopic Olfactory Receptor, Suppresses Oxidative Stress-Induced Senescence in Human Keratinocytes

doi: 10.3390/antiox11112180

Figure Lengend Snippet: Proposed role of OR2AT4 in the suppression of senescence in human keratinocytes. Sandalore binds and activates OR2AT4 to increase the levels of second messengers, mainly intracellular calcium. The increased intracellular calcium level activates the CAMKKβ/AMPK/mTORC1/autophagy signaling axis. OR2AT4 inhibits p21, a marker of cellular senescence, by activating AMPK phosphorylation. Overall, OR2AT4 activation by sandalore may improve cellular senescence in human keratinocytes.

Article Snippet: The primary antibodies used for immunoblotting were as follows: OR2AT4 (ab105828, 36 kDa; Abcam, Cambridge, MA, USA), GNAL (sc-48345, 46 kDa; Santa Cruz Biotechnology, Santa Cruz, CA, USA), adenylate cyclase 3 (ADCY3) (sc-588, 130 kDa; Santa Cruz Biotechnology), p21/CIP1/CDKN1A (NBP2-294563, 21 kDa; Novus, St. Louis, MO, USA), phosphorylated calcium/calmodulin-dependent protein kinase kinase (p-CaMKKβ) (12818S, 50 kDa; Cell Signaling Technology, Danvers, MA, USA), CaMKKβ (sc-50341, 50 kDa; Santa Cruz Biotechnology), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) (44-1150G, 62 kDa; Invitrogen, Carlsbad, CA, USA), AMPK (sc-25792, 62 kDa; Santa Cruz Biotechnology), LC3 antibody (sc-0376404, 15 kDa; Santa Cruz Biotechnology), phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1) antibody (ab63552, 289 kDa; Abcam), mTORC1 antibody (2983S, 289 kDa; Cell Signaling Technology), phosphorylated p70 ribosomal S6 kinase (p-p70S6K) antibody (2211S, 80 kDa; Cell Signaling Technology), p70S6K antibody (2217S, 80 kDa; Cell Signaling Technology), and β-actin (sc-47778, 43 kDa; Santa Cruz Biotechnology).

Techniques: Marker, Phospho-proteomics, Activation Assay

Journal: Frontiers in Bioscience-Landmark

Article Title: Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction

doi: 10.31083/fbl25565

Figure Lengend Snippet: Fig. 4. Trimetazidine regulates the expression of AMPK signaling pathway during myocardial I/R. The expressions of adenosine

Article Snippet: The primary antibodies used in this study included endothelial nitric oxide synthase (eNOS) (1:1000, 27120-1-AP,Wuhan, China), phosphorylated eNOS (p-eNOS) (1:1500, AF3247, Affinity, Jiangsu, China), endothelin-1 (ET-1) (1:1000, 12191- 1-AP, Proteintech, Wuhan, China), ZO-1 (1:5000, 21773- 1-AP, Proteintech, Wuhan, China), Occludin (1:1000, #91131, CST, Danvers, MA, USA), VE-cadherin (1:1000, 27956-1-AP, Proteintech, Wuhan, China), AMPK (1:2000, 10929-2AP, Proteintech, Wuhan, China), phosphorylated Adenosine Monophosphate (AMP)-activated protein kinase (p-AMPK) (1:1500, AP1002, ABclonal, Wuhan, China), kruppel-like factor 4 (KLF4) (1:1000, ab241666, Abcam, Cambridge, MA, USA), peroxisome proliferatoractivated receptor delta (PPARδ) (1:1000, 60193-1-lg, Proteintech, Wuhan, China), CD31 (1:1000, #77699, CST, Danvers, MA,USA), vascular endothelial growth factor (VEGF) (1:1500, AF5131, Affinity, Jiangsu, China), protein kinase B (AKT) (1:3000, 10176-2-AP, Proteintech, Wuhan, China), phosphorylated AKT (p-AKT) (1:2000, 66444-1-lg, Proteintech, Wuhan, China), phosphatidylinositol 3-Kinase (PI3K) (1:5000, 60225-1-lg, Proteintech, Wuhan, China), phosphorylated-PI3K (p-PI3K) (1:1000, AF4372, Affinity, Jiangsu, China) and GAPDH (1:1500, AF7021, Affinity, Jiangsu, China).

Techniques: Expressing

Journal: Frontiers in Bioscience-Landmark

Article Title: Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction

doi: 10.31083/fbl25565

Figure Lengend Snippet: Fig. 6. Effect of trimetazidine with varying concentrations on AMPK signaling pathway in primary mouse heart microvascular

Article Snippet: The primary antibodies used in this study included endothelial nitric oxide synthase (eNOS) (1:1000, 27120-1-AP,Wuhan, China), phosphorylated eNOS (p-eNOS) (1:1500, AF3247, Affinity, Jiangsu, China), endothelin-1 (ET-1) (1:1000, 12191- 1-AP, Proteintech, Wuhan, China), ZO-1 (1:5000, 21773- 1-AP, Proteintech, Wuhan, China), Occludin (1:1000, #91131, CST, Danvers, MA, USA), VE-cadherin (1:1000, 27956-1-AP, Proteintech, Wuhan, China), AMPK (1:2000, 10929-2AP, Proteintech, Wuhan, China), phosphorylated Adenosine Monophosphate (AMP)-activated protein kinase (p-AMPK) (1:1500, AP1002, ABclonal, Wuhan, China), kruppel-like factor 4 (KLF4) (1:1000, ab241666, Abcam, Cambridge, MA, USA), peroxisome proliferatoractivated receptor delta (PPARδ) (1:1000, 60193-1-lg, Proteintech, Wuhan, China), CD31 (1:1000, #77699, CST, Danvers, MA,USA), vascular endothelial growth factor (VEGF) (1:1500, AF5131, Affinity, Jiangsu, China), protein kinase B (AKT) (1:3000, 10176-2-AP, Proteintech, Wuhan, China), phosphorylated AKT (p-AKT) (1:2000, 66444-1-lg, Proteintech, Wuhan, China), phosphatidylinositol 3-Kinase (PI3K) (1:5000, 60225-1-lg, Proteintech, Wuhan, China), phosphorylated-PI3K (p-PI3K) (1:1000, AF4372, Affinity, Jiangsu, China) and GAPDH (1:1500, AF7021, Affinity, Jiangsu, China).

Techniques:

Journal: Frontiers in Bioscience-Landmark

Article Title: Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction

doi: 10.31083/fbl25565

Figure Lengend Snippet: Fig. 8. Trimetazidine alleviates functional impairment of OGD/R-induced primary mouse heart microvascular endothelial cells through AMPK signaling pathway. (A) The expressions of eNOS, p-eNOS and ET-1 were detected by western blot. (B) The NO

Article Snippet: The primary antibodies used in this study included endothelial nitric oxide synthase (eNOS) (1:1000, 27120-1-AP,Wuhan, China), phosphorylated eNOS (p-eNOS) (1:1500, AF3247, Affinity, Jiangsu, China), endothelin-1 (ET-1) (1:1000, 12191- 1-AP, Proteintech, Wuhan, China), ZO-1 (1:5000, 21773- 1-AP, Proteintech, Wuhan, China), Occludin (1:1000, #91131, CST, Danvers, MA, USA), VE-cadherin (1:1000, 27956-1-AP, Proteintech, Wuhan, China), AMPK (1:2000, 10929-2AP, Proteintech, Wuhan, China), phosphorylated Adenosine Monophosphate (AMP)-activated protein kinase (p-AMPK) (1:1500, AP1002, ABclonal, Wuhan, China), kruppel-like factor 4 (KLF4) (1:1000, ab241666, Abcam, Cambridge, MA, USA), peroxisome proliferatoractivated receptor delta (PPARδ) (1:1000, 60193-1-lg, Proteintech, Wuhan, China), CD31 (1:1000, #77699, CST, Danvers, MA,USA), vascular endothelial growth factor (VEGF) (1:1500, AF5131, Affinity, Jiangsu, China), protein kinase B (AKT) (1:3000, 10176-2-AP, Proteintech, Wuhan, China), phosphorylated AKT (p-AKT) (1:2000, 66444-1-lg, Proteintech, Wuhan, China), phosphatidylinositol 3-Kinase (PI3K) (1:5000, 60225-1-lg, Proteintech, Wuhan, China), phosphorylated-PI3K (p-PI3K) (1:1000, AF4372, Affinity, Jiangsu, China) and GAPDH (1:1500, AF7021, Affinity, Jiangsu, China).

Techniques: Functional Assay, Western Blot

Journal: Frontiers in Bioscience-Landmark

Article Title: Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction

doi: 10.31083/fbl25565

Figure Lengend Snippet: Fig. 9. Trimetazidine alleviates barrier damage of OGD/R-induced primary mouse heart microvascular endothelial cells through AMPK signaling pathway. (A) The endothelial cell permeability experiment was used to detect the cell permeability (scale bar: 50

Article Snippet: The primary antibodies used in this study included endothelial nitric oxide synthase (eNOS) (1:1000, 27120-1-AP,Wuhan, China), phosphorylated eNOS (p-eNOS) (1:1500, AF3247, Affinity, Jiangsu, China), endothelin-1 (ET-1) (1:1000, 12191- 1-AP, Proteintech, Wuhan, China), ZO-1 (1:5000, 21773- 1-AP, Proteintech, Wuhan, China), Occludin (1:1000, #91131, CST, Danvers, MA, USA), VE-cadherin (1:1000, 27956-1-AP, Proteintech, Wuhan, China), AMPK (1:2000, 10929-2AP, Proteintech, Wuhan, China), phosphorylated Adenosine Monophosphate (AMP)-activated protein kinase (p-AMPK) (1:1500, AP1002, ABclonal, Wuhan, China), kruppel-like factor 4 (KLF4) (1:1000, ab241666, Abcam, Cambridge, MA, USA), peroxisome proliferatoractivated receptor delta (PPARδ) (1:1000, 60193-1-lg, Proteintech, Wuhan, China), CD31 (1:1000, #77699, CST, Danvers, MA,USA), vascular endothelial growth factor (VEGF) (1:1500, AF5131, Affinity, Jiangsu, China), protein kinase B (AKT) (1:3000, 10176-2-AP, Proteintech, Wuhan, China), phosphorylated AKT (p-AKT) (1:2000, 66444-1-lg, Proteintech, Wuhan, China), phosphatidylinositol 3-Kinase (PI3K) (1:5000, 60225-1-lg, Proteintech, Wuhan, China), phosphorylated-PI3K (p-PI3K) (1:1000, AF4372, Affinity, Jiangsu, China) and GAPDH (1:1500, AF7021, Affinity, Jiangsu, China).

Techniques: Permeability

Journal: Nutrients

Article Title: β-Hydroxy-β-methyl Butyrate Regulates the Lipid Metabolism, Mitochondrial Function, and Fat Browning of Adipocytes

doi: 10.3390/nu15112550

Figure Lengend Snippet: The effects of HMB on the expression of proteins relating to lipid metabolism in 3T3-L1 adipocytes (n = 3). Black circles and red squares represent the data obtained without and with HMB treatment, respectively. HMB (50 µM) was added to the differentiation medium for 48 h. T-AMPK level was used as a control for p-AMPK level.; T-Sirt1 level was used as a control for p-Sirt1; GAPDH level was used as a control for other protein levels. Values are means, with their standard errors represented by vertical bars. Significance levels were marked as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 via two-tailed t -test.

Article Snippet: The primary antibodies used were the following: CCAAT/enhancer-binding protein alpha (C/EBPα, 18311-1-AP, Proteintech, Chicago, IL, USA, 1:500); peroxisome proliferator-activated receptor gamma (PPARγ, 2435S, CST, Boston, MA, USA, 1:1000); phosphorylated (p)-AMP-activated protein kinase AMPK (p-AMPK, 2535S, CST, Boston, MA, USA, 1:1000); phosphorylated (p)-silent information regulator 2 related enzyme 1 (p-Sirt1, 2314S, CST, Boston, MA, USA, 1:2000); hormone-sensitive lipase (HSL, 4107S, CST, Boston, MA, USA, 1:1000); lipoprotein lipase (LPL, ab137821, Abcam, Cambridge, Cambs, UK, 1:1000); uncoupled protein 3 (UCP3, 10750-1-AP, Proteintech, Chicago, IL, USA, 1:500); PGC-1α (ab54481, Proteintech, Chicago, IL, USA, 1:3000); PRDM16 (ab106410, Abcam, Cambridge, Cambs, UK, 2 μg/mL); UCP1 (14670, CST, Boston, MA, USA, 1:1000); glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004-1-Ig, Proteintech, Chicago, IL, USA, 1:7000).

Techniques: Expressing, Control, Two Tailed Test