Preparation of tissue and cell extracts Visceral epidydimal fat pads and livers were harvested, washed in ice cold saline and dissected rapidly into 3 5 mg pieces, followed by pre incubation for this 20 minutes in Krebs Ringer HEPES Buffer at 95%O2 5%CO2 at 37 C. Samples were maintained at 95%O2 5% CO2 at 37 C for the duration of the Inhibitors,Modulators,Libraries experiments. Sam ples from individual mice were each assayed as separate experimental groups so that a given PAR2 stimulated AMPK response could be attributed to a single mouse. For cell culture studies, cells were used at 80% conflu ence and complete media was exchanged for serum free media 2 hours prior to the experiments. Tissues or cells were treated with or without 100nM of 2fAP at 37 C and then homogenized in ice cold lysis buffer containing TBS pH 7.
4 supplemented with 1 mM EGTA, with pro teinase and phosphatase inhibitors and either 1%Triton X 100 and 0. 1% SDS or 1% NP 40, followed by cen trifugation Inhibitors,Modulators,Libraries for 10 min at 14,000 rpm at 4 C. Adenine Nucleotide Measurement Cilengitide by LC MS MS NIH3T3 cells were incubated with 100 nM of 2f AP for 0 2 hrs, washed in cold PBS and 5% of perchloric acid was added to the cells. Acid insoluble material was removed by centrifu gation, and perchloric acid was extracted from the supernatant by three washes with 10% excess of a 1,1 mixture of tri n octylamine and 1,1,2 trichlorotrifluoroethane. The nucleotide mixture was subjected to online LC ESI MS MS analysis using an Agilent 1100 capillary HPLC pump interfaced with an LCQ Deca XP ion trap mass spectrometer. The mass spectrometer was set up for monitoring the fragmentation of the ions of AMP and ATP.
A 0. 5 �� 250 mm Zorbax SB C18 column was used, and the flow rate was 8. 0 uL min. A 5 min gradient of 0 20% methanol in 400 mM 1,1,1,3,3,3 hexa fluoro 2 propanol, followed by a 25 min gradient of 20 50% methanol in 400 mM HFIP was employed for the separation. Inhibitors,Modulators,Libraries The ratio of AMP over ATP was calculated by comparing the integrated Inhibitors,Modulators,Libraries areas of AMP to ATP from selected ion chromatograms with the considera tion of the differences in ionization and fragmentation efficiencies of the two nucleotides. Protein analysis and immunoblotting Lysates from cells and liver or fat tissue were subjected to 8% SDS PAGE gel and transfer to PVDFfl membranes, followed by Western blotting with the following antibodies, Rabbit polyclonal anti phosphor AMPK Thr172 or anti phosphor ACC Ser79, mouse mono clonal anti AMPKa1 2, rabbit anti Flag, rabbit anti b arrestin or rabbit anti CAMKKb.
Blots were imaged inhibitor with Alexa680 con jugated rabbit and IR800 conjugated mouse secondary antibodies using the LICOR Odyssey imaging system, and LICOR soft ware was used to calculate integrated intensities of bands, phospho AMPK and ACC levels were normalized total AMPK and actin respectively.