Figure 3.

Immunoblot and in vitro thioester formation assays. (A,B) Immunoblot assay of UBA5 mutants in UBA5^−/− HEK293T cells. Indicated constructs (0.1 µg for UBA5 or UBA5 mutant, 0.5 µg for UFC1 or UFC mutant, and 2 µg for UFM1ΔC2) were expressed in UBA5-deficient HEK293T cells. Twenty-four hours after transfection, the cell lysates were subjected to immunoblot analysis with indicated antibodies. Bar graphs indicate the quantitative densitometric analyses of FLAG-UBA5-MYC-UFM1ΔC2 and FLAG-UFC1-MYC-UFM1ΔC2 intermediates relative to free FLAG-UBA5 and FLAG-UFC1, respectively. Statistical analyses were performed using the unpaired t-test (Welch test). The data represents the means ± SE of three separate experiments. (**) P < 0.01. (C,D) In vitro thioester formation assay of UFM1 by UBA5 (C) and of UFM1 by UFC1 (D). The assay was conducted as described in Methods. Data shown are representative of three separate experiments.