total t ampk  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Tomatine Improves Glucose Metabolism and Mitochondrial Respiration in Insulin-Resistant Hepatocyte Cell Lines AML12 and HepG2 via an AMP-Activated Protein Kinase-Dependent Pathway.

doi: 10.3390/cells14050329

Figure Lengend Snippet: Figure 3. Effects of tomatine on AMPK/PGC1α signaling in IR hepatocytes. AML12 or HepG2 cells were cultured under conditions designed to induce IR, as described in Section 2. (A,B) Western blot analysis was performed for p-AMPK (Thr172), AMPK, PGC1α, and GAPDH. (C,D) The bar graphs represent the quantitative expression of p-AMPK (Thr172)/t-AMPK and PGC1α/GAPDH. (E) The mRNA expression of Pgc1α was analyzed using RT-qPCR. Data are presented as the mean ± SD (n ≥3). Significant differences (p < 0.05) were determined using one-way ANOVA followed by Tukey’s post hoc test. *,# p < 0.05, **,## p < 0.001, and ***,### p < 0.0001. “ns” indicates nonsignifi- cant differences.

Article Snippet: Antibodies against phospho(p)-AMPK (Thr 172, #2531; Cell Signaling, Danvers, MA, USA), total(t)-AMPK (#2793; Cell Signaling), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; 66369-1-Ig; Proteintech, Chicago, IL, USA), total OXPHOS cocktail (#ab110413, Abcam, Cambridge, MA, USA), VDAC (#4866; Cell Signaling), and GAPDH (#2118; Cell Signaling) were used for Western blot analysis.

Techniques: Cell Culture, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cells

Article Title: Tomatine Improves Glucose Metabolism and Mitochondrial Respiration in Insulin-Resistant Hepatocyte Cell Lines AML12 and HepG2 via an AMP-Activated Protein Kinase-Dependent Pathway.

doi: 10.3390/cells14050329

Figure Lengend Snippet: Figure 5. Effects of tomatine on the activation of AMPK in IR hepatocytes via an AMPK-dependent pathway. HepG2 cells were cultured under conditions designed to induce IR, as described in Section 2. The cells were transfected with either nontargeting siRNA (siCtrl) or AMPK-directed siRNA (siAMPK) for 24 h and then exposed to high glucose and insulin for 24 h, with or without tomatine (1 µM). (A) Western blot analysis was performed for p-AMPK (Thr172), AMPK, and GAPDH. (B) The quantitative bar graphs of p-AMPK (Thr172)/GAPDH are presented. (C) The mRNA expression of Pgc1a was analyzed using RT-qPCR. Data are presented as the mean ± SD (n ≥3). Significant differences (p < 0.05) were determined using one-way ANOVA followed by Tukey’s post hoc test. # p < 0.05, ## p < 0.001, and ### p < 0.0001.

Article Snippet: Antibodies against phospho(p)-AMPK (Thr 172, #2531; Cell Signaling, Danvers, MA, USA), total(t)-AMPK (#2793; Cell Signaling), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; 66369-1-Ig; Proteintech, Chicago, IL, USA), total OXPHOS cocktail (#ab110413, Abcam, Cambridge, MA, USA), VDAC (#4866; Cell Signaling), and GAPDH (#2118; Cell Signaling) were used for Western blot analysis.

Techniques: Activation Assay, Cell Culture, Transfection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cells

Article Title: Tomatine Improves Glucose Metabolism and Mitochondrial Respiration in Insulin-Resistant Hepatocyte Cell Lines AML12 and HepG2 via an AMP-Activated Protein Kinase-Dependent Pathway.

doi: 10.3390/cells14050329

Figure Lengend Snippet: Figure 7. Effects of tomatine on mitochondrial oxidative function in IR hepatocytes via an AMPK- dependent pathway. AML12 or HepG2 cells were cultured under conditions designed to induce IR, as described in Section 2. The cells were transfected with either nontargeting siRNA (siCtrl) or AMPK-directed siRNA (siAMPK) for 24 h and then exposed to high glucose and insulin for 24 h, with or without tomatine (1 µM). (A) The OCR was measured as described in Section 2 and normalized to cell numbers. (B) Total OCR was calculated and normalized to the protein content of each group. (C) Intracellular ATP levels were measured. (D,E) The subunits of the OXPHOS complex were analyzed using SDS-PAGE followed by immunoblotting, with densitometric quantification provided. Data are shown as the mean ± SD (n ≥3). Significant differences (p < 0.05) were determined using one-way ANOVA followed by Tukey’s post hoc test. # p < 0.05, ## p < 0.001, and ### p < 0.0001. “ns” indicates nonsignificant differences. * Complex IV subunit (with a theoretical molecular weight of 38 kDa) was not detected.

Article Snippet: Antibodies against phospho(p)-AMPK (Thr 172, #2531; Cell Signaling, Danvers, MA, USA), total(t)-AMPK (#2793; Cell Signaling), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; 66369-1-Ig; Proteintech, Chicago, IL, USA), total OXPHOS cocktail (#ab110413, Abcam, Cambridge, MA, USA), VDAC (#4866; Cell Signaling), and GAPDH (#2118; Cell Signaling) were used for Western blot analysis.

Techniques: Cell Culture, Transfection, SDS Page, Western Blot, Molecular Weight

Journal: Journal of Diabetes Research

Article Title: Enhancement of Mitochondrial Function by the Neurogenic Molecule NSI-189 Accompanies Reversal of Peripheral Neuropathy and Memory Impairment in a Rat Model of Type 2 Diabetes

doi: 10.1155/2022/8566970

Figure Lengend Snippet: AMPK activity in DRG tissues from ZDF animals: lumbar DRG tissues were obtained from control (Ctrl), ZDF, and NSI-treated ZDF (ZDF+NSI) rats and underwent (a) Western blotting or (c) cytochrome c oxidase activity assay. (a) Phosphorylated AMPK (pAMPK) and total ERK (T-ERK) Western blot image. (b) Intensity of bands shown in (a). (c) Activity of cytochrome c oxidase (Cox IV). Data are mean ± SEM of N = 7-8 for Western blotting and N = 4 for cytochrome c oxidase activity assay; ∗ p < 0.05, analyzed by one-way ANOVA with Holm-Sidak's post hoc test. Data are mean ± SEM.

Article Snippet: Blots were incubated with antibodies to phosphorylated (Thr172) AMPK (pAMPK; 1 : 500, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and total AMPK (T-AMPK; 1 : 500, Cell Signaling Technology, Boston, MA).

Techniques: Activity Assay, Western Blot

Journal: Journal of Diabetes Research

Article Title: Enhancement of Mitochondrial Function by the Neurogenic Molecule NSI-189 Accompanies Reversal of Peripheral Neuropathy and Memory Impairment in a Rat Model of Type 2 Diabetes

doi: 10.1155/2022/8566970

Figure Lengend Snippet: AMPK activity in brain cortex tissues from ZDF animals. The cortex was obtained from control (Ctrl), ZDF, and NSI-treated ZDF (ZDF+NSI) rats and underwent Western blotting for (a) pAMPK and T-ERK signals. (b) Intensity of bands shown in (a). Data are mean ± SEM of N = 6.

Article Snippet: Blots were incubated with antibodies to phosphorylated (Thr172) AMPK (pAMPK; 1 : 500, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and total AMPK (T-AMPK; 1 : 500, Cell Signaling Technology, Boston, MA).

Techniques: Activity Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Protective effect of Astragaloside IV on chronic intermittent hypoxia-induced vascular endothelial dysfunction through the calpain-1/SIRT1/AMPK signaling pathway

doi: 10.3389/fphar.2022.920977

Figure Lengend Snippet: Effects of As-IV and Capn1 knockout on the SIRT1/AMPK/eNOS signaling pathway in CIH-induced mice. (A–F) Western blot analysis of calpain-1, SIRT1, Thr 172 AMPK phosphorylation, t-AMPK, Ser 1177 eNOS phosphorylation and eNOS protein expression in all groups with quantification ( n = 3). (G–J) Representative fluorescence images and fluorescence intensity of calpain-1 and SIRT1 in all groups of aortic sections (×400 magnification; n = 3). Calpain-1 (green), SIRT1 (red), Hoechst 33342 (blue). Data are presented as means ± SD. #P < 0.05, vs. Con group, ** p < 0.01, *P < 0.05, vs. CIH group.

Article Snippet: Ser 1177 eNOS phosphorylation, total endothelial nitric oxide (eNOS), Thr 172 AMPK phosphorylation, total 5’ AMP activated protein kinase (t-AMPK), vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1) were obtained from ABclonal (Wuhan, China).

Techniques: Knock-Out, Western Blot, Expressing, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Protective effect of Astragaloside IV on chronic intermittent hypoxia-induced vascular endothelial dysfunction through the calpain-1/SIRT1/AMPK signaling pathway

doi: 10.3389/fphar.2022.920977

Figure Lengend Snippet: Effects of As-IV, MDL-28170 and SRT1720 administration on calpain-1, SIRT1, AMPK, and eNOS protein expression in HCAECs. (A–F) The protein expression levels of calpain-1, SIRT1, Thr 172 AMPK phosphorylation, t-AMPK, Ser 1177 eNOS phosphorylation and eNOS were analyzed using Western blots ( n = 3). Data are presented as means ± SD. ** p < 0.01, *P < 0.05, vs. CIH group.

Article Snippet: Ser 1177 eNOS phosphorylation, total endothelial nitric oxide (eNOS), Thr 172 AMPK phosphorylation, total 5’ AMP activated protein kinase (t-AMPK), vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1) were obtained from ABclonal (Wuhan, China).

Techniques: Expressing, Western Blot