Western Blot Analysis of Cell Signaling

The detailed procedure was reported previously64 (link). Briefly, protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein from whole cell lysates were solubilized in SDS-sample buffers and separated on SDS polyacrylamide gels. Membranes were incubated with antibodies against MUC1, p65, p50, the phosphor and total AMPKα. The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Beverly, MA, USA). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millpore, Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (Bio-Rad, Hercules, CA, USA).