Figure 2

ERK activity profile is not affected by PCPTP1 depletion. A) RNAi-mediated knockdown of endogenous PCPTP1 in PC12 cells. PC12 cells stably expressing siRNA targeted to nucleotides 2380–2398 in PCPTP1 cDNA (pSR-2380) were generated via retroviral transduction. Empty vector (pSR) was used as control and PCPTP1 knockdown was rescued by transduction of pSR-2380-containing cells with a retroviral expression construct for the mouse ortholog PTPBR7. Equal amounts of protein from the resulting stable cell pools were subjected to immunoprecipitation using α-SL antiserum and captured proteins were analyzed on Western blots using monoclonal antibody 6A6. B) Transient and sustained ERK1/2 signaling in wild type PC12 cells. After overnight serum deprivation, cells were stimulated with 100 ng/ml EGF (upper panels) or 50 ng/ml NGF (lower panels) for the indicated time points. Cell lysates were subjected to Western blot analysis and phospho-ERK1/2 signals (pERK1, pERK2) were detected using a chemiluminescence imaging system. The same blot was reprobed with total ERK1 antibody to correct for loading differences. C) Transient ERK activity is not affected in PCPTP1 knockdown cells. Serum-starved cells were stimulated with 10 ng/ml EGF for 2 or 5 min, respectively, and harvested. Relative phospho-ERK1/2 levels in the protein lysates, determined as above, are presented as mean values ± SEM from three independent experiments. D) PCPTP1 knockdown in PC12 cells does not significantly alter ERK activity at reduced levels of EGF. Serum starved cells were stimulated with 0.075 ng/ml EGF for the indicated time points and relative phospho-ERK1/2 levels were determined as before. Results are mean values ± SEM (n = 4).