Mammalian Cell Immunoblot and IP

Whole-cell lysates of mammalian cells and colon tissues were prepared and analyzed for immunoblot as previously performed [14 (link)]. For IP, cells were washed twice in cold PBS and lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) plus phosphatase and protease inhibitors (Roche Applied Science, Mannheim, Germany). Whole-cell extracts were incubated with the appropriate primary antibodies overnight at 4 °C. Antibody-bound proteins were precipitated with protein A/G beads according to the manufacturer’s protocol. The beads were washed four times with lysis buffer and then eluted in 2x SDS sample loading buffer. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), transferred to PVDF membranes (Merck Millipore), and detected using appropriate primary antibodies coupled with a horseradish peroxidase-conjugated secondary antibody using chemiluminescence (Thermo Fisher Scientific, MA, USA) and the LAS-4000 imager (GE Healthcare Life Sciences, Piscataway, NJ, USA).