rabbit anti phospho ampkα 1  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression

doi: 10.1038/srep19781

Figure Lengend Snippet: ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P < 0.001). AU: Arbitrary Unit. ( B ) representative western blots of C2C12 cells and relative levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in C2C12 myoblast transfected with pCMV6-Entry-HSPD1 vector (pcDNA3.1 was used as a negative control). 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control and TR45 was used as positive control. Data are presented as the means ± SD. # significantly different from pCMV6-Entry-HSPD1 (P < 0.0001). AU: Arbitrary Unit. *significant results. ( C ) relative expression levels of 4 HNE, p-AMPKα, p-AMPKα/(AMPKα1 + AMPKα2) in the soleus . Open bars, sedentary mice; shaded bars, trained mice. AU: Arbitrary Unit.

Article Snippet: Next, the membrane was further incubated in a primary antibody, anti-Hsp60 (diluted 1:1,000, mouse monoclonal antibody ab13532, Abcam, Cambridge, UK), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) diluted 1:3,000, rabbit polyclonal antibody ADI905784, Enzo Life Sciences, Inc. NY, USA), or anti-PGC1α (diluted, 1:1,000, mouse monoclonal antibody ST1202, Calbiochem, Inc. San Diego, CA, USA), or anti-Hsp70 (diluted, 1:1,000, mouse monoclonal antibody C92F3A-5, Santa Cruz Biotechnologies), or anti-Alix (diluted, 1:500, mouse monoclonal antibody sc-53538, Santa Cruz Biotechnologies), or anti-Mn SOD (diluted 1:1000, rabbit polyclonal antibody ADI-SOD-110, Enzo Life Sciences), or anti- AMPKα1 (diluted 1:1000, rabbit polyclonal antibody 07-350SP, Millipore, Temecula, CA, USA), or anti-AMPKα2 (diluted, 1:1000, rabbit polyclonal antibody 07-363SP, Millipore), or anti-phospho-AMPKα (diluted, 1:1000, rabbit polyclonal antibody 07-681SP, Millipore), or anti-TFAM (diluted 1:1000, rabbit polyclonal antibody DR1071, Calbiochem, Inc. San Diego, CA, USA), or anti-4 HNE (diluted 1:1000, rabbit polyclonal antibody, ab46545, Abcam).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Positive Control

Journal:

Article Title: Green and black tea extracts inhibit HMG-CoA reductase and activate AMP-kinase to decrease cholesterol synthesis in hepatoma cells

doi: 10.1016/j.jnutbio.2008.07.011

Figure Lengend Snippet: Phosphorylation of AMP-kinase by tea extract. Phosphorylated AMP-kinase was measured by immunoquantitation with an antibody specific for the phosphorylated enzyme. A, Enzyme phosphorylation in hepatoma cells after a 3-h treatment with green (grey bars) or black (black bars) tea extract; each data set included an untreated sample and samples treated with the AMP-kinase activators AICAR or metformin (1 mM each). Values represent the mean and standard deviation of 2 experiments; values that are statistically different from untreated controls are indicated with asterisks. The immunoblots below the graph illustrate the increase in AMP-kinase phosphorylation with each treatment (GTE, green tea extract; BTE, black tea extract). B, Enzyme phosphorylation in hepatoma cells at various times after treatment with 15 μg/ml of green (circles) or black (boxes) tea extract. Values represent the mean and standard deviation of 4 experiments; closed symbols are statistically different from untreated controls. Statistical significance was determined by one-way analysis of variance with Dunnett's post-hoc text, p < 0.05.

Article Snippet: The membrane was blocked with 0.05% Tween-20 and 5% defatted milk, and then incubated in this same buffer with rabbit antibody to total AMP-kinase (Anti-AMPK α-pan, 1:2000; Upstate USA, Inc., Charlottesville, VA) or to phosphorylated AMP-kinase (Anti-phospho-AMPKα, 1:500; Upstate USA, Inc.) overnight at 4°C.

Techniques: Standard Deviation, Western Blot