Figure 3

NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75^NTR, p75^NTR CTF, and α-tubulin as a loading control. As described [40], NGF induced the proteolysis of p75^NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, _**P < 0.01. (D) PC12-ApoER2 was treated as previously described and then incubated with 100 ng/mL NGF for different times (0–240 min). Cell lysates were used for protein detection by western blot analysis. AKT phosphorylation is observed immediately after the addition of NGF. as well as the apparition of p75NTR CTF. α-tubulin is shown as a loading control. (E) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments.