Figure 7. Knockdown of Sprouty4 potentiates MAPK activation and PC12 cell differentiation in response to NGF.

A) Spry4 mRNA levels were analyzed by real-time PCR in PC12 cells transfected with scrambled (control, Ctrl) or Spry4 shRNA constructs. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing scrambled or Spry4 shRNA constructs. Quantitative analysis is shown as averages SD of triplicate determinations. The levels of Spry4 mRNA were normalized using the expression of the housekeeping gene Tbp. *p<0.001 (Student's t test). B) Spry4 protein levels were analyzed by IB in Cos cells transfected with Spry4 shRNA or a control vector together with Myc-Spry4. Actin is shown as loading control. C) Spry4 knockdown on Erk2/MAPK activation was analyzed in PC12 cells treated with NGF (50 ng/ml) for 10 min. Erk2/MAPK activation was evaluated by transient transfection of HA-tagged Erk2 plasmid with a control or Spry4 shRNA construct into PC12 cells. Erk2 activation (P-Erk2) was evaluated by HA-immunoprecipitation (IP) followed by IB with a specific antibody that recognizes the phosphorylated forms of ERK/MAPK. Reprobing of the same blot with anti-HA antibody is shown. Numbers below the lanes are normalized to the levels of HA-Erk2. D) Morphological differentiation of PC12 cells transfected with scramble (Ctrl) or Spry4 shRNA cloned in the retroviral vector pGFP-V-RS and treated with NGF (50 ng/ml) for 24 h. E) The histogram shows the quantification of the relative number of GFP positive neurite-bearing cells longer than 1 or 2 cell diameters in the different experimental conditions. The results are shown as averages SD of a representative experiment performed in quadruplicates. *p<0.001 (Student's t test).