Figure 3.

Hu proteins act independently of the NGF/Ras cascade. (A–E) HuC-induced neuronal phenotype of PC12 cells is not accompanied by ERK activation. Expression of activated-ERK in PC12 cells overexpressing HuC in the absence of additional NGF (A–C) or cells expressing GFP-Myc in the presence of additional NGF (D and E) 9 d after transfection. There was no detectable expression of activated ERK in neuron-like PC12 cells that overexpressed HuC. Bar = 5 μm. (F) HuC-R3 cannot prevent NGF-induced PC12 differentiation. PC12 cells transfected with HuC-R3, H-Ras^N17 (dominant negative) or FLAG-GFP (negative control) were cultured in medium containing additional NGF. Transfected cells were selected by using G418. Morphology of transfected cells was evaluated 9 d after transfection same as in Fig. 3. Although H-Ras^N17 misexpression almost completely blocked NGF-induced differentiation, HuC-R3 misexpression showed no significant influence on differentiation. (G) Dominant-negative H-Ras cannot prevent HuC-induced neuronal phenotype. H-Ras^N17 and HuC, or H-Ras^V12(dominant active) and HuC-R3 were cotransfected into PC12 cells. Morphology of the cells expressing both proteins was evaluated as in the legend for Fig. 2. Dominant-negative H-Ras (H-Ras^N17) could not suppress HuC-induced neuronal phenotype, and HuC-R3 could not suppress dominant active Ras (H-Ras^V12)-induced differentiation.