Figure 9. Generation of CAR CTF1 precedes CTF2 production.

(A) Stable cell lines of U87-MG and U251N expressing either the V5 tag alone (mock) or CAR with a C-terminal V5 tag were generated. Equal amounts of cell lysates were analyzed by SDS-PAGE and Western blot using a mouse monoclonal antibody raised against the V5 tag. Full-length CAR, CTF1 and CTF2 were detected similarly to lysates of U87 CAR cells probed with anti-CAR C-term. antibody RP291 (Figure 8A). (B) U251N V5 and U251N CAR-V5 cells were treated overnight with MG132 (25 µM) or DMSO vehicle control. Equal amounts of proteins from cell lysates were used for anti-V5 Western blots. In the case of the MG132-treated cells, CTF1 levels accumulated while CTF2 nearly disappeared, similar to previous experiments with the U87 CAR cell line (Figure 8B). (C) MEF wild-type (MEF WT) or PS 1- and 2-knockout MEF cells (MEF PS1/2 KO) were infected with lentivirus to express full-length CAR with a C-terminal V5 tag. Cells were lysed 3 days post-infection and lysates were analyzed by Western blot using antibody raised against the V5 tag. MEF WT cells, but not MEF PS1/2 KO cells, contained CAR CTF2, indicating that presenilin is required for generation of the 14 kDa CTF2 fragment of CAR. (D) Verification of knockdown in ADAM10 expression in U87 CAR-V5 cells using ADAM10 shRNA (#6675); shown are anti-ADAM10 and anti-GAPDH Western blots. Lysates were also analyzed by Western blot using antibody raised against the V5 tag. The ADAM10 shRNA stable cell line had a decreased CAR CTF1 level, as expected. CAR CTF2 levels also decreased, indicating that shedding is a prerequisite for RIP of CAR.