Rabbit anti phospho ampk thr172 antibody

The samples were prepared as described previously [33 (link)]. Protein concentrations were quantified by the BCA Protein Assay Kit (Thermo, Rockford, USA). Samples containing equal amounts of protein were subjected to SDS-PAGE in a Bio-Rad miniature slab gel apparatus and electrophoretically transferred onto a nitrocellulose membrane. The membranes were incubated with 5 % BSA/PBST for 1 h at 25 °C, and incubated overnight at 4 °C with primary antibodies and 1 h at 25 °C with secondary antibodies respectively. Cell Signaling Technology Inc (Danvers, Mass) was the supplier for the following primary antibodies: rabbit polyclonal anti-phospho-eNOS (Ser1177) antibody, rabbit anti-phospho-AMPK (Thr172) antibody, rabbit anti-AMPK antibody. Mouse anti-eNOS monoclonal antibody was obtained from BD Transduction Laboratories (Lexington, Ky).

HUVECs were lysed using cell lysis buffer [10 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% (v/v) Triton-X 100, 1× Protease inhibitor cocktail (Roche, Basel, Switzerland), 1× PhosSTOP phosphatase inhibitor cocktail (Roche, Basel, Switzerland)] for 30 min at 4°C, and then centrifuged at 15,000×g for 1 min. The equal amount of cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transfer to immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) as previous described [22 (link)]. The rabbit anti-phospho-AMPK (Thr172) antibody, rabbit anti-AMPK antibody, rabbit anti-COX-2 antibody, rabbit anti-ICAM-1 antibody and rabbit anti-VCAM-1 antibody were purchased from Cell signaling technology (Danvers, MA, USA). The rabbit anti-APRT antibody was purchased from Abcam (Cambridge, UK) and the mouse anti-β-actin antibody was purchased from Novus biologicals (Littleton, CO, USA). After the membranes were incubated with 1^st antibodies at 4°C overnight followed by the corresponding 2^nd antibody for 1 h at room temperature (RT), immunoreactive bands were detected by chemiluminescence (VisGlowTM, Visual Protein, Taipei, TW) and recorded using Kodak XAR-5 film (Rochester, NY, USA). The detected signals were scanned and then quantified using Image J software (http://image.nih.gov/ij/).

Protein extraction and western blot analysis were performed as reported from our previous publication [12 (link)]. The antibodies used were as follows: mouse anti-Pax7 (1 : 500, DSHB AB_528428), rabbit anti-MyoD (1 : 500, Santa Cruz sc-760), mouse anti-MyHC (1 : 500, DSHB MF20), rabbit anti-Tom20 (1 : 1000, Cell Signaling 42406), mouse anti-Sirt1 (1 : 1000, Cell Signaling 8469), rabbit anti-acH3 (1 : 1000, Cell Signaling 9649), rabbit anti-phospho AMPK (Thr172) antibody (1 : 1000, Cell Signaling 2535), rabbit anti-AMPK (1 : 1000, Cell Signaling 2603), rabbit anti-phospho P70S6K (Thr421/Ser424) (1 : 1000, Cell Signaling 9204), rabbit anti-P70S6K (1 : 1000, Cell Signaling 9202), rabbit anti-phospho RPS6 (Ser240/244) antibody (1 : 1000, Cell Signaling 2215), rabbit anti-RPS6 (1 : 1000, Cell Signaling 2217), and rabbit anti-tubulin antibody (1 : 500, Santa Cruz sc-9104). Densitometric analysis was performed using ImageQuant. Normalization of phosphorylated and total proteins was performed by using tubulin or vinculin. Finally, the ratio between the phosphorylated and total protein was indicated.