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. 2022 Jun 8;21(7):100256. doi: 10.1016/j.mcpro.2022.100256

Fig. 6.

Fig. 6

Comparison of biotin ligase activity. Human embryonic kidney (HEK) cells were transfected with several biotin ligases including BioID2, MicroID2, lbMicroID2 (containing additional I120A, N125A mutants), miniTurbo, and TurboID. Constructs were either expressed alone (A and B) or conjugated to the transmembrane protein MARCH1 (C). At 24 to 48 h post-transfection, cells were labeled with biotin for 1 h (A and C) or for 10 min (B), and cell lysates were harvested. HA-tagged biotin ligase expression was determined using anti-HA antibody. Streptavidin–HRP labels biotinylated proteins in the whole-cell supernatant. Below, densitometry of streptavidin normalized to HA expression with unconjugated (A and B) and the MARCH1-conjugated biotin ligases (C). A–C, n = 3 experiments, ∗p < 0.05, ∗∗p < 0.01 versus BioID2 by ANOVA. HA, hemagglutinin; HRP, horseradish peroxidase.