Figure 7. Excessive PIP₃ accumulation and mTORC1 activation in cells expressing mutant GP130.

(A–C) Formation of PIP₃ at the cell membrane was monitored as an indicator of PI3K activity by immunofluorescent staining using the PIP₃-specific GST-GRP1PH probe. The representative images show (A) naive 293T cells or (B) cells transiently expressing the indicated chimeric EpoR/GP130 receptor. Cells were stimulated for the indicated time with hyper–IL-6 (HypIL6; 100 ng/ml) or Epo (50 U/ml). (C) Images were then used for cell segmentation analysis and quantification with MetaMorph software. For each time point a total of 150 to 300 cells was analyzed, and results are depicted as mean ± SEM (*P < 0.05 when compared with unstimulated isogenic cells). Scale bar: 10 μm. Also refer to Supplemental Figure 9. (D and E) Immunoblot analysis of (D) naive 293T cells or (E) cells transiently expressing the EpoR/gp130^F2 receptor. Cultures were serum starved and, where indicated, exposed to the PI3K inhibitor LY294002 (25 μM) 60 minutes prior to stimulation with hyper–IL-6 (100 ng/ml) or Epo (50 U/ml) for the indicated period. (F) Immunoblot analysis of 293T cells transiently expressing the indicated mutant EpoR/GP130 receptor. Serum-starved cells were stimulated for 60 minutes with Epo (50 U/ml). The schematic shows the transfected EpoR/GP130 receptors, which encode tyrosine (Y) to phenylalanine (F) substitution or truncation mutations in the cytosolic domain of human GP130. The consensus function of the individual Y residues and their amino acid position are depicted. Also refer to Supplemental Figure 11. Note that the pERK1/2 lanes were run on the same gel but were noncontiguous.