Figure 3. CPXV012 blocks ATP binding to TAP1 and TAP2.

(A) CPXV012 inhibits ATP binding to TAP. TAP1, TAP2, and ^C8CPXV012 were coexpressed in Sf9 cells as indicated. TAP1/TAP2 or TAP1/TAP2/^C8CPXV012 complexes were affinity purified using anti-TAP1 (mAb 148.3) or anti-C8 antibodies, respectively. Dynabead immobilized proteins were pre-incubated with 15 µM 8-azido-ATP[γ]biotin in the presence or absence of 5 mM ATP for 5 min on ice and subsequently subjected to UV irradiation. Dynabead immobilized proteins were then separated by SDS-PAGE and detected by immunoblotting with the antibodies as indicated. Biotinylated proteins were visualized using extravidin-HRP conjugate. In the presence (open bars) and absence of ^C8CPXV012 (filled bars), the amount of ATP cross-linked TAP was normalized to the TAP2 protein expression levels. (B) CPXV012 inhibits ATP binding sites to TAP1 and TAP2. TAP1, ^TsnTAP2, and ^flagCPXV012 were coexpressed and crude membranes were incubated with 8-azido-ATP[γ]biotin (15 µM) in the presence or absence of ATP (5 mM) for 5 min on ice. After UV irradiation, proteins were immunoprecipitated with TAP1 (mAb 148.3) or flag-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Immunoprecipitated samples were then separated by SDS-PAGE and analyzed by immunoblotting with extravidin-HRP or the corresponding antibodies. The amount of TAP photo cross-linked by 8-azido-ATP in the absence (filled bars) or presence (open bars) of ^flagCPXV012 was normalized to TAP1 or ^TsnTAP2 protein expression levels, respectively.