china anti p ampk cell signaling technology 2535s massachusetts  (Cell Signaling Technology Inc)

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of starvation on protein synthesis and degradation. Cells were incubated in starvation medium for 1, 5, or 24 h. (A) The relative mRNA expression of Atg1 and Murf1 was quantified by reverse transcription-quantitative PCR. (B and C) Protein phosphorylation levels of AMPK and p70S6K, and the protein expression ratio of LC3II/LC3I after (B) 5, or (C) 24 h of starvation were analyzed by western blotting. Representative blot images are shown in the upper part. Values are expressed as fold-change compared with the values of control cells incubated in regular DMEM. Values are presented as the mean ± SEM (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control. Atg1, atrogin-1; Con, control; Murf1, muscle ring finger 1; p-, phosphorylated; Stv, starvation.

Article Snippet: The membranes were blocked with Blocking One-P (Nacalai Tesque) for 60 min at room temperature (22–24°C) and then incubated overnight (16–18 h) at 4°C with primary antibodies against each of the following proteins: LC3 (#2775), 70-kDa ribosomal protein S6 kinase (p70S6K; #9202), phosphorylated-p70S6K (#9205), AMP-activated protein kinase (AMPK; #2532), and phosphorylated-AMPK (#2535), all purchased from Cell Signaling (Danvers, MA, USA), or β-actin (#sc47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Incubation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of single nutrient supplementation on protein metabolism and morphological atrophy. Cells were incubated in starvation medium with (colored bars) or without (black bars) the indicated nutrients for (A) 5 or (B and C) 24 h. (A) Relative mRNA expression of Atg1 and Murf1 was quantified by reverse transcription-quantitative PCR. (B) Protein phosphorylation levels of AMPK and p70S6K, and the protein expression ratio of LC3II/LC3I were analyzed by western blotting. Representative blot images are shown on the left side. (C) Representative fluorescence images of MHC antibody-stained myotubes at ×200 magnification were captured using a fluorescence microscope (BZ-X700; Keyence). Values are expressed as fold-change compared with the values of the control cells incubated in regular DMEM. Values are presented as mean ± SEM (n=3-4). Δ P<0.1, *P<0.05, **P<0.01, ***P<0.001 vs. starved cells. Atg1, atrogin-1; Con, control; Murf1, muscle ring finger 1; p-, phosphorylated; Stv, starvation; Glc, glucose; Gln, glutamine; LA, lactate; βOHB, β-hydroxy butyric acid; αKG, α-ketoglutarate; Leu, leucine.

Article Snippet: The membranes were blocked with Blocking One-P (Nacalai Tesque) for 60 min at room temperature (22–24°C) and then incubated overnight (16–18 h) at 4°C with primary antibodies against each of the following proteins: LC3 (#2775), 70-kDa ribosomal protein S6 kinase (p70S6K; #9202), phosphorylated-p70S6K (#9205), AMP-activated protein kinase (AMPK; #2532), and phosphorylated-AMPK (#2535), all purchased from Cell Signaling (Danvers, MA, USA), or β-actin (#sc47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Incubation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Fluorescence, Staining, Microscopy, Control