Figure 7.

Initial NGF signaling through ERK and Akt pathways is unaffected by NPCD knock-down. PC12 cells were transfected with either scrambled (Sc) or NPCD siRNAs for 3 d before stimulation with NGF (100 ng/ml). Activation of signaling proteins was assessed 15 min and 1 h after NGF stimulation by Western blot with phospho-specific antibodies. We tested activation of ERKs 1 and 2 (row 1), the ERK target p90RSK (row 2), Akt (row 3), and the Akt target S6 ribosomal protein (row 4). No consistent effects on activation of any of these proteins at either time point was observed (a slight decrease seen here at 15 min for phosphoS6 was not seen in other experiments). NPCD knock-down of the 55 kDa doublet (row 5) and the 41 kDa band (row 6) were monitored in the same lysates; knock-down by NPCD siRNAs was clearly evident (85% knock-down compared with scrambled, 55 kDa; 60% knock-down, 41 kDa). GAPDH Western blotting was used in these blots as a loading control (row 7). This experiment was repeated twice with similar results. RNAi, RNA interference.