FIGURE 4.

RvE1 regulates the phosphorylation of ribosomal protein S6. A, whole cell lysates were derived from CHO-hChemR23 cells treated with or without RvE1 (10 nm, 15 min, 37 °C) and pulled down with a mouse antibody to ribosomal protein S6 Ab. Immunoprecipitates (∼70 μg/lane) were blotted with the phospho-Akt substrate antibody (pAkt(S) Ab; lanes c and d). rS6 was detected with rS6 Ab (lanes g and h) to confirm protein loading. IB, immunoblot; IP, immunoprecipitation. B, immunofluorescence microscopy of rS6 phosphorylation. CHO-hChemR23 cells and CHO-mock cells were incubated with or without RvE1 (10 nm, 37 °C, 15 min). Cells were stained with anti-phospho-rS6 (Ser²³⁵/Ser²³⁶) Ab followed by Alexa Fluor® 568 secondary Ab (red) to determine the distribution of phosphorylated rS6 within the cells. Actin filaments were labeled with Alexa Fluor® 488-phalloidin (green), and nuclei were labeled with DAPI (blue) as described under “Experimental Procedures.” C, reduction of RvE1-enhanced p30 phosphorylation by pharmacological inhibitors. CHO-hChemR23 cells were incubated with wortmannin (200 nm), PD98059 (50 μm), or SB203580 (10 μm) for 15 min. Cells were then exposed to RvE1 (10 nm, 37 °C, 15 min) and stained with anti-phospho-rS6 (Ser²³⁵/Ser²³⁶) Ab using Alexa Fluor® 568 secondary Ab (red) to determine the phosphorylation of rS6. Actin filaments were labeled with Alexa Fluor® 488-phalloidin (green), and nuclei were labeled with DAPI (blue).