Figure 5. Pertussis toxin treatment and transient expression of β-ARK blocks Pcp2-stimulated differentiation in NGF-treated PC12 cells.

(A) Effects of pertussis toxin on NGF-stimulated neurite growth in PC12 cells +/−Pcp2 expression. Pertussis toxin (PTX; 150 nM) was added to culture medium at the time of media change to +/−Dox. The medium was changed daily and neurite lengths quantified at 72 h of NGF treatment. Results are the means±range of two independent experiments each with 30–60 cells for each condition. (B) Pcp2-PC12 cells were transiently transfected with GFP+β-ARK or vector (pcDNA3) as described in the Experimental section. Images of Pcp2-Pc12 cells in −Dox medium transfected with β-ARK+GFP and imaged after 72 h of NGF stimulation. Phase contrast (A) image shows five cells; two of which are expressing β-ARK (denoted with arrows; B). (C) Background fluorescence in non-transfected cells under these conditions. Scale bar=20 μM. (C) Quantified neurite length/cell in vector and β-ARK transfected Pcp2-PC12 cells (all NGF-treated for 72 h) in the presence and absence of Pcp2 expression (+/−Dox). Results are the means±S.D. of three experiments where 30–60 cells were counted in each experiment. (D) Ras activity was measured in β-ARK and vector transfected PC12 cells as described in the Experimental section. After overnight serum starvation, cells were treated with NGF for 5 and 15 min and Ras activity was determined on 150 μg of cell lysate. Total Ras from each condition (30 μg) is shown below. (E) Relative Ras activity for β-ARK and vector transfected cells (n=5 at t=0, n=3 at 5, 15 min) was observed and it is expressed as means±S.E.M.