Western blot, immunofluorescence and immunohistochemical analyses Cells have been lysed in lysis buffer containing a protease inhibitor and phenyl methyl sulfonyl fluoride. Equal amounts of every sample had been 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 resolution at room temperature for 1 h, followed by overnight incubation with various major antibodies. Antibodies against AMPK B1, AMPK, phospho AMPK, P70S6K, phospho P70S6K, AKT, phospho AKT, mTOR, and phospho mTOR were bought from Cell Signaling, whereas antibodies against JNK, phospho JNK, ERK and phospho ERK were purchased from Santa Cruz Biotechnology, Inc.
The blots have been then incubated with goat anti rabbit or anti mouse secondary antibodies that had been conjugated to horseradish peroxidase and visualized through an enhanced chemiluminescence method. B Actin was applied because the loading control. For immunofluorescence pop over here evaluation, SKOV3 cells were cultured on cover slips and transiently transfected with AMPK B1 expressing plasmid. The preparation and examination of pEGFP AMPK B1 transfected cells have been performed as previously described. Immunohistochemical staining for AMPK B1 was performed on an ovarian cancer tissue array, and an antibody against AMPK B1 was made use of to examine the expression of AMPK B1. Procedures and the scoring of outcomes have been performed as previously described, and the examination of immunohistochemical staining was performed by two independent observers.
Confocal microscopy The cellular localization of AMPK B1 was examined in A2780CP and SKOV3 read this post here cells just after the transient expression of the pCMV6 AMPK B1 GFP tagged plasmid . The analytical procedure was reported previously, and fluorescence signals had been captured applying confocal microscopy. Cell proliferation assay The cell proliferation assay was performed applying a cell proliferation kit, and information were obtained from three separate experiments that were performed in triplicate. Clonogenic assay Approximately 800 cells have been plated in triplicate in six properly plates to kind colonies for as much as 2 4 weeks, along with the medium was replaced each 3 7 days. The colonies had been then stained with crystal violet and counted. Anchorage independent development assay in soft agar A soft agar colony formation assay was made use of to identify the capacity of ovarian cancer cells to undergo anchorage independent cell development upon various treatment options.
Sterile 2% and 0. 6% agarose gel stocks in 2? MEM containing 20% FBS were ready, and single cell suspensions have been prepared by suspending 1000 cells in two ml of complete medium containing 0. 3% agar. The cell suspensions were plated on major of a solidified bottom layer with 1% agar within the complete medium, and also the plates had been incubated at 37 C within a humidified incubator for 14 21 days.