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.
Shipley et al. BMC Microbiology 2005 5:48 doi:10.1186/1471-2180-5-48