Journal: Cells
Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.
doi: 10.3390/cells14040294
Figure Lengend Snippet: Figure 5. AMPK activation with sodium salicylate mediates c-Myc ubiquitinylation and nuclear depletion. (A) Exposure of HCT116 colon cancer cells to 1 or 3 mM salicylate for 48 h (lanes 2 and 3, respectively) elicited the loss of c-Myc expression accompanied by CIP2A, but no change in the levels of S62 phosphorylated c-Myc. Cells were also treated with a combination of salicylate (1 or 3 mM) and compound C (dorsomorphin 100 nM) for 8 h (lanes 5 and 6, respectively). (B) Salicylate induced phosphorylation of PP2A Y307. Samples were analyzed with an anti-pY307-PP2Ac antibody, and the blots were reprobed with an anti-PP2Ac antibody. (C) Sodium salicylate induced AMPK-mediated ubiquitinylation of c-Myc. HEK293 cells were transiently transfected with c-Myc-HA (lanes 1 through 4) or a mock construct (HA-vector, lane 5). Cells were then either left untreated (lanes 1 and 5) or treated with compound C (lane 2), sodium salicylate (lane 3), or a combination of both (lane 4), for three hours. C-Myc-HA-tagged protein was then immunopurified and its ubiquitination levels analyzed with a ubiquitin antibody by western blot (upper panel, ubiquitin). Immunoblots of the corresponding cell lysates (ubiquitin antibody) and purified proteins (HA antibody) are shown (lower
Article Snippet: The mouse monoclonal antibody against CIP2A (2G103B5) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
Techniques: Activation Assay, Expressing, Phospho-proteomics, Transfection, Construct, Plasmid Preparation, Ubiquitin Proteomics, Western Blot, Purification