Figure 3

v-H-ras induces ERK-dependent CSE1L phosphorylation and secretion. (A) Immunoblot analysis of CSE1L phosphorylation with synthesized CSE1L protein produced by wheat germ cell-free protein synthesis system. Lane1: B16-dEV cell lysate; lane 2: CSE1L-depleted cell lysates produced by depleting CSE1L with anti-CSE1L antibodies and A/G plus agarose; lane 3: recombinant GST-CSE1L protein; lane 4: CSE1L protein. (B) Levels of threonine-phosphorylated CSE1L in B16-dEV and B16-Ras cells treated with DMSO or PD98059 for 24 h. The levels were analyzed by immunoprecipitation of the samples with anti-CSE1L antibodies and immunoblotting with HRP-conjugated anti-phosphothreonine antibodies. The immunoblot was reprobed with anti-CSE1L antibodies. Control immunoprecipitation was performed by using mouse anti-GFP antibodies. (C) Levels of secretory threonine-phosphorylated CSE1L in cell number-standardized conditioned media harvested from serum-starved B16-dEV and B16-Ras cells treated with DMSO or PD98059 for 24 h. The levels were analyzed by immunoprecipitation of the samples with anti-CSE1L antibodies and immunoblotting with HRP-conjugated anti-phosphothreonine antibodies.