Figure 1

Presence of hyper-, hypo-, and non-phosphorylated CSE1L in tumor cells as analyzed by antibodies against CSE1L and phosphorylated CSE1L. a The levels of phosphorylated and non-phosphorylated CSE1L in B16-dEV, B16-Ras, and PD98059-treated B16-Ras cells were analyzed with anti-CSE1L antibody (clone 3D8) as indicated. b The levels of phospho-CSE1L and phospho-ERK1/2 in B16-dEV, B16-CSE1L, and B16-Ras cells were analyzed with anti-phospho-CSE1L and anti-phospho-ERK1/2 antibodies as indicated. c Presence of hyper- and hypo-phosphorylated CSE1L in B16-Ras cells analyzed using lambda protein phosphatase. B16-Ras cell lysates treated with or without lambda protein phosphatase were subjected to immunoblotting with anti-CSE1L (3D8), anti-phospho-CSE1L, and anti-phospho-ERK1/2 antibodies as indicated. d Presence of non-phosphorylated and phosphorylated CSE1L as analyzed using serum-starved and serum re-fed non-cancerous cell lines. The phosphorylation of CSE1L in cell lysates from serum starved or serum starved and serum retreated HT-29 colorectal cancer cells, human foreskin fibroblast cells, NIH3T3 cells, and B16F10 melanoma cells were analyzed using the anti-CSE1L (clone 24) antibody. e Distribution of secretory phospho-CSE1L in the extracellular secretion vesicles (arrowhead) surrounding B16-Ras cells was analyzed by immunofluorescence with anti-phospho-CSE1L antibodies. Each immunoblot was repeated at least three times and showed similar results. The data shown here are the representative immunoblots. β-actin levels were assayed as a control. pp-CSE1L hyper-phosphorylated CSE1L, p-CSE1L hypo-phosphorylated CSE1L, non-p-CSE1L non-phosphorylated CSE1L.