Fig. 4.

ERα^S167 mediates IGF-induced phosphorylation, transcription, and growth. A, ERα-negative C4-12 cells were transiently transfected (48 h) with empty vector (Vector), ERα-WT, point-mutated ERα^S118A (S118A), or ERα^S167A (S167A) and serum starved overnight. Cells were then exposed to IGF or E2 (1 h) and immunoblotted against the proteins of interest. ERα (anti-ERα) was used to ensure equal transfection rates, and MAPK (anti-MAPK) served as a loading control. B, C4-12 cells were transfected with empty vector (Vector), ERα-WT or a mutant form of ERα (ERα-S118A or ERα-S167A), and anchorage-independent growth in response to IGF or E2 was assessed by soft agar assay. Colony-forming ability was expressed as fold change over vector-transfected cells. C, IGF1R, IRS1, and TFF1 gene expression of C4-12 cells transfected with empty vector, ERα-WT, or ERα-S167A was measured by RT-qPCR. Target gene expression was normalized to RPLP0 gene expression and presented as fold change over vector-transfected control. All error bars depict sd and all results are representative of at least three independent replicates.