total ampk #2532s antibody  (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