Activation of PKC does not affect APP internalization. HEK-695 cells were pre-treated with TAPI-1 in serum free DMEM for 1 hour prior to biotinylation. After biotinylation and quenching, cells were incubated at 37°C for various time periods in the presence of TAPI-1 and either DMSO or PMA (1 μM). The medium was collected and biotinylated sAPPα was isolated and analyzed by immunoblot using 6E10 antibodies. The cells were stripped and lysed, and biotinylated APP was isolated and assessed by immunoblot analysis with antibodies to the APP C-terminal. A. Internalization of biotinylated APP was nearly absent in cells that were biotinylated and stripped (B/S) while still on ice. NB, non-biotinylated; B, biotinylated, not stripped. B. PMA, in the presence of TAPI-1, did not affect release of biotinylated sAPPα(upper panel), or internalization of full-length biotinylated APP (middle panel). C. Bands depicting biotinylated and internalized APP were quantitated by densitometry, normalized, and expressed as means ± SEM from 3 experiments. D. In the absence of TAPI-1, PMA caused a marked increase in release of endogenous and transfected sAPPα from HEK-695 cells.
Carey et al. BMC Cell Biology 2005 6:30 doi:10.1186/1471-2121-6-30