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. Author manuscript; available in PMC: 2017 Oct 17.

Published in final edited form as: J Alzheimers Dis. 2017;56(4):1263–1269. doi: 10.3233/JAD-161094

(A) Overexpression of PS1N and PS1Cs did not restore γ-secretase activity in Pen-2^-/- cells. All cells were transfected with NotchΔE. In Pen-2^-/- cells, in addition to NotchΔE, PS1N and PS1C were also co-expressed. Expression of PS1C was detected by PS1C-specific antibody #5643 (top panel). PS1N was detected by anti-PS1N antibody (second panel). Expression of NotchΔE was detected by anti-Myc antibody (third panel). NICD was detected by antibody #4147 (fourth panel). The immunoblot in the fourth panel was also probed with anti-GAPDH to indicate relative loading of samples (bottom panel). (B) The association of PS1N and PS1C was not disturbed by the deletion of Pen-2. Pen-2^-/- cells were transfected with PS1N along with one of the PS1C variants (PS1C_293, PS1C_296, PS1C_299, PS1C_334, PS1C₃₄₆). Cell lysates were immunoprecipitated with anti-PS1N and anti-PS1C antibodies. Up panel, immunoprecipitates were probed with anti-PS1C antibody #5643. Low panel, immunoblots were probed with anti-PS1N. WT cell and Wt-7 cell were used as controls. (C) Pen-2 plays a crucial role in Notch binding to PS1. All cells were transfected with NotchΔE in combination with PS1wt or both PS1N and PS1C as indicated. After cultured in the presence or absence of γ-secretase inhibitor compound E (CompE) for 24 hrs, cell lysates were subjected to immunoprecipitation using antibodies against Pen-2, PS1C, and Myc, respectively as indicated. First panel, immunoblots were probed with anti-Myc to determine the full-length NotchΔE. Second panel, immunoprecipitates were probed with anti-NICD to detect NICD. Third panel, immunoblots were probed with anti-PS1C #5643 antibody to detect all PS1Cs of different lengths; Bottom panel, immunoblots were probed with anti-Pen-2 antibody. Note that anti-Pen2 immunoprecipitates (second panel, lanes 1, 4, 7, 10, and 13) non-specifically react with antibody #4147, which is specific to NICD.

(A) Overexpression of PS1N and PS1Cs did not restore γ-secretase activity in Pen-2^-/- cells. All cells were transfected with NotchΔE. In Pen-2^-/- cells, in addition to NotchΔE, PS1N and PS1C were also co-expressed. Expression of PS1C was detected by PS1C-specific antibody #5643 (top panel). PS1N was detected by anti-PS1N antibody (second panel). Expression of NotchΔE was detected by anti-Myc antibody (third panel). NICD was detected by antibody #4147 (fourth panel). The immunoblot in the fourth panel was also probed with anti-GAPDH to indicate relative loading of samples (bottom panel). (B) The association of PS1N and PS1C was not disturbed by the deletion of Pen-2. Pen-2^-/- cells were transfected with PS1N along with one of the PS1C variants (PS1C_293, PS1C_296, PS1C_299, PS1C_334, PS1C₃₄₆). Cell lysates were immunoprecipitated with anti-PS1N and anti-PS1C antibodies. Up panel, immunoprecipitates were probed with anti-PS1C antibody #5643. Low panel, immunoblots were probed with anti-PS1N. WT cell and Wt-7 cell were used as controls. (C) Pen-2 plays a crucial role in Notch binding to PS1. All cells were transfected with NotchΔE in combination with PS1wt or both PS1N and PS1C as indicated. After cultured in the presence or absence of γ-secretase inhibitor compound E (CompE) for 24 hrs, cell lysates were subjected to immunoprecipitation using antibodies against Pen-2, PS1C, and Myc, respectively as indicated. First panel, immunoblots were probed with anti-Myc to determine the full-length NotchΔE. Second panel, immunoprecipitates were probed with anti-NICD to detect NICD. Third panel, immunoblots were probed with anti-PS1C #5643 antibody to detect all PS1Cs of different lengths; Bottom panel, immunoblots were probed with anti-Pen-2 antibody. Note that anti-Pen2 immunoprecipitates (second panel, lanes 1, 4, 7, 10, and 13) non-specifically react with antibody #4147, which is specific to NICD.