Western Blot Protein Detection Protocol

Western blots were performed as described previously [33 (link), 50 (link)]. Briefly, cells were lysed with Triton Lysis buffer, and protease and phosphatase inhibitors (Sigma-Aldrich) were added. From each sample, 15 μg of protein was separated on a NuPAGE 4–12% acrylamide gel (Invitrogen). The following antibodies were used for Western blotting: rabbit anti-KLF4 antibody [49 (link)] at 1:10,000 dilution; rabbit anti-RHOF antibody (LSBio LS-C353833) at 1:500 dilution; rabbit anti-phospho-p65 (Cell Signaling, S536) at 1:1,000 dilution; rabbit anti-p65 (Cell Signaling, C22B4) at 1:1,000 dilution; mouse anti-IKK2 (Cell Signaling) at 1:1,000 dilution; mouse anti-β-actin at 1:10,000 dilution; rabbit anti-GAPDH (Cell Signaling) at 1:10,000 dilution; and mouse anti-α-tubulin at 1:15,000 dilution as described previously [50 (link)].