Figure 3

Absence of N-terminal fragment and confirmation that 55 kDa fragment is derived from APP. (A) Staining with antibody to N-terminal APP. SHSY5Y human neuroblastoma cells were either untreated (cont) or infected with HSV1, then incubated for a further 6 h. Cell lysates were analysed by Western blotting using an anti-N-terminal APP antibody 22C11. No evidence for increase in an N-terminal fragment of the expected M Wt. was observed, suggesting the latter may be metabolised further or secreted. (B) Strengthening of 55 kDa band in uninfected APP-transfected SHSY5Y cells. Normal SHSY5Y human neuroblastoma cells, mock-transfected SHSY5Y cells (mTf) or APP695 transfected cells (APP Tf) were uninfected prior to harvest. In addition normal SHSY5Y cells which had been infected with HSV1 (lane labelled HSV1) for 6 h were also prepared, to allow position of the HSV1-senstive 55 kDa band to be clearly identified. Cell lysates were subjected to Western blot analysis, and probed with an anti-C-terminal APP antibody (Sigma Aldrich A8717) at 1:4000 dilution. Arrows indicate the position of the band intensified after HSV1 infection. The intensity of full-length APP bands was appreciably greater than those in the non-transfected cells. In addition the intensity of several smaller bands was also greater than the intensity of the same bands in the mock-transfected (mTf) or normal SHSY5Y (cont), including a band at the same position as the band intensified on HSV1 infection of normal SHSY5Y cells (marked by the arrows), suggested this band is genuinely derived from APP.