FIGURE 7.

Unnatural amino acid incorporation into PDCD5. A, scheme of unnatural amino acid UV cross-linking using BzF. B, BzF incorporation into Amber codon variants of PDCD5 and the G protein β-subunit. HEK-293T cells were transfected with the indicated FLAG-PDCD5 constructs, BzF tRNA and BzF amino acid synthetase, and treated with 1 mm BzF 3 h post-transfection. Lysates were immunoblotted to determine the level of expression of the variants. The percent incorporation was measured as the ratio of the truncated protein band, resulting from a lack of BzF incorporation into the Amber codon, to the full-length protein band, resulting from BzF incorporation into the Amber codon. PDCD5 averaged 6% incorporation compared with 75% for Gβ₁.