Figure 3.

Pretreatment with TGFβ induces GDNF responsiveness, but does not affect expression of the GDNF receptors. (A) 100,000 CG neurons per well were plated and allowed to attach for 2 h. The cells were treated with 2 ng/ml TGFβ for 3 h or left untreated as indicated. Subsequently no factor or GDNF (10 ng/ml) or GDNF (10 ng/ml) plus TGFβ (2 ng/ml) were added for 30 min before harvesting of the cells in Laemmli sample buffer. (B) CG neurons were handled as in (A) except that PD98059 or Wortmannin was added to prove specific inhibition of ERK- and Akt-phosphorylation by the respective inhibitors. (C) Cells were pretreated for 3 h with TGFβ (2 ng/ml), thereafter an antibody blocking all three TGFβ-isoforms (anti-pan TGFβ) was added where indicated. 10 min later GDNF (10 ng/ml), TGFβ (2 ng/ml) or both factors were added for 30 min and the cells subsequently harvested in Laemmli sample buffer. Protein extracts (A–C) were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes and probed with anti– phospho ERK-, anti–phospho Akt- or anti–GFRα1-antibodies. To control for equal loading the membrane stripped and probed with an antibody against nonphosphorylated ERK. (D) CG neurons were differently treated to investigate the requirement of persistent TGFβ or GDNF treatment for survival. Cells were pretreated for 3 h with TGFβ3 (2 ng/ml). Thereafter, either TGFβ was blocked by addition of a pan TGFβ antibody and GDNF (10 ng/ml) was added or only GDNF was added in the absence of the antibody. Similarly the neurons were pretreated with GDNF (10 ng/ml) for 3 h and after a washout of GDNF, TGFβ3 (2 ng/ml) was added. As controls, neurons were simultaneously treated with GDNF and TGFβ in the presence or absence of the pan TGFβ antibody. The surviving neurons were counted after 24 h, each bar represents the mean and standard deviation of two independent experiments with every condition tested in triplicate per assay (***, P ≤ 0.01). (E) RT-PCR of the GDNF-receptors GFRα1 and Ret and immunoblot of GFRα1 using total RNA and protein extracts, respectively, from CG neurons treated for 3 h with TGFβ or untreated controls. For the immunoblot Neuro2a cells stably expressing GFRα1 were used as positive control. (F) Anti phospho-ERK immunoblot with extracts from CG neurons, pretreated with TGFβ for the indicated time points and subsequently treated with GDNF for 30 min or left untreated. As control for equal loading an immunoblot with an antibody detecting nonphosphorylated ERK was used.