Equal amounts of each sample were fractionated by SDS PAGE and electroblotted onto an Immobilon P Membrane. The membrane was blocked with 5% non fat dry milk in a TBS with Tween solution at room temperature for 1 h, followed by overnight incubation with various primary antibodies. Antibodies against AMPK B1, AMPK, phospho AMPK, P70S6K, phospho P70S6K, contain AKT, phospho AKT, mTOR, and phospho mTOR were purchased from Cell Signaling, whereas antibodies against JNK, phospho JNK, ERK and phospho ERK were purchased from Santa Cruz Biotechnology, Inc. The blots were then incubated with goat anti rabbit or anti mouse secondary Inhibitors,Modulators,Libraries antibodies that were conjugated to horseradish peroxidase and visualized via an enhanced chemiluminescence system. B Actin was used as the loading control.
For immunofluorescence analysis, SKOV3 cells were cultured on cover slips and transiently transfected with AMPK B1 expressing plasmid. The preparation and exam ination of pEGFP AMPK B1 transfected cells were per formed as previously described. Immunohistochemical staining for AMPK B1 was performed on an ovarian cancer tissue array, and an antibody Inhibitors,Modulators,Libraries against AMPK B1 was used to examine the expression of AM PK B1. Procedures and the scoring of results were per formed as previously described, and the examin ation of immunohistochemical staining was performed Inhibitors,Modulators,Libraries by two independent observers. Confocal microscopy The cellular localization of AMPK B1 was examined in A2780CP and SKOV3 cells after the transient expression of the pCMV6 AMPK B1 GFP tagged plasmid.
The analytical procedure was reported pre viously, and fluorescence signals were captured using confocal microscopy. Cell proliferation assay The cell proliferation Inhibitors,Modulators,Libraries assay was performed using a cell proliferation kit, and data were obtained from three separate experiments that were performed in triplicate. Clonogenic assay Approximately 800 cells were plated in triplicate in 6 well plates to form colonies for up to 2 4 weeks, and the medium was replaced every 3 7 days. The colonies were then stained with crystal violet and counted. Anchorage independent Inhibitors,Modulators,Libraries growth assay A soft agar colony formation assay was used to determine the capacity of ovarian cancer cells to undergo anchorage independent cell growth upon different treatments. Sterile 2% and 0. 6% agarose gel stocks in 2�� MEM containing 20% FBS were prepared, and single cell suspen sions were prepared by suspending 1000 cells in 2 ml of full medium containing 0.
3% agar. The cell suspensions were plated on top of a solidified bottom layer with 1% agar in the full medium, and the plates were incubated at 37 C in a humidified incubator for 14 21 days. The col onies were then counted using a dissecting microscope. Flow cytometry The DNA content, cell selleck chem inhibitor cycle distribution and percentage of apoptotic cells of each sample were assessed by flow cytometry. Cells were cultured in 6 well plates, and float ing and attached cells were harvested by trypsinization, centrifuged and resuspended in PBS.