Supersignal west femto maxmum sensitivity substrate

Cells were exposed to Myc inhibitors for the indicated periods of time and then harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. Equivalent amounts of protein were subjected to SDS-PAGE and immunoblotting according to previously-described procedures [12 (link), 13 (link)]. Antibodies used included mouse monoclonal antibodies (mAbs) against AMPK (1:1000 dilution: catalog no. 2532, Cell Signaling Technology, Beverly, MA) and Myc (1:500 dilution: Clone 9E10, Santa Cruz Biotechnology, Inc. Santa Cruz, CA) a rabbit polyclonal antibody against AMPK phospho-Thr₁₇₂ (1:1000 dilution, catalog no. 2535, Cell Signaling Technologies). As a control for protein loading, blots were also probed with a mouse mAb against β−actin (1:10,000 dilution: cat. no.3700S Cell Signaling Technology). Immunoblots were developed using an enhanced chemiluminescence reagent according to the directions of the supplier (SuperSignal West Femto Maxmum Sensitivity Substrate, Thermo Fisher Scientific, Inc. Waltham, MA).

After extraction with a mixture of T-PER Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA), Complete Mini (Roche, Basel, Switzerland) and PhosSTOP (Roche) from cells, lysates were fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfected to nitrocellulose membranes (Millipore, Billerica, MA, USA). A 5% milk in phosphate buffered saline was used to block the non-specific binding sites for one hour. The membranes were then incubated at 4 °C for overnight with the following primary antibodies: XRCC6 (Proteintech, Chicago, IL, USA. 1:500), β-catenin (total) (Proteintech, 1:500), β-catenin (actived) (Bioworld Technology, St. Louis Park, MN, USA, 1:500), c-MYC (Proteintech, 1:500), Cyclin D1 (Proteintech, 1:500) or β-actin (Sigma-Aldrich, 1:20,000). The secondary antibodies were anti-rabbit IgG (Sigma-Aldrich, 1:5000) or anti-mouse IgG (Sigma-Aldrich, 1:5000). SuperSignal West Femto Maxmum Sensitivity Substrate (Thermo Fisher Scientific) was used to perform the chemiluminescence detection of the blots. Images were analyzed with ImageJ software by loading the image as a grayscale picture.