Acsl4

Cell pellets were lysed in RIPA buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS (supplemented with 1 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM DTT, and 0.1 M PMSF) for 20 minutes on ice and centrifuged at high speed for 20 minutes at 4 °C. Extracts containing equal quantities of proteins, determined by using the BCA Protein Assay Kit (Pierce) were separated by SDS-polyacrylamide gel electrophoresis (12.5% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were incubated in blocking buffer [5% (w/v) non-fat dry milk dissolved in PBST (1X PBS containing 0.05% [v/v] Tween 20)] for 1 hour at room temperature. Membranes were then incubated with antibodies to phosphorylated ERK (Cell Signaling), E-Cadherin (Cell Signaling), ACSL4 (Thermo), or Actin (Abcam) diluted 1:1000 in blocking buffer for 1–2 hours at room temperature. Secondary antibodies conjugated to horse-radish peroxidase were obtained from Biorad and used at a dilution of 1:10,000. Bound antibodies were detected using enhanced chemiluminescence (Biorad).