Fig. 3.

Co-translational formation of the extraordinarily stable FIH-OTUB1 HD. (A) Following ectopic expression of the indicated proteins, the FIH-OTUB1 heterodimer (HD) was allowed to form for 24 h prior to the analysis of the HD stability in hypoxia when FIH is inhibited and no additional HD can be formed. (B) HD formation kinetics and (C) HD formation during translation inhibition by cycloheximide (CHX) according to the experimental setups described in Fig. S3B. Re-Ox CHX, addition of CHX at the same time as re-oxygenation was started; Re-Ox CHX pre, pre-incubation of cells with CHX for 1 h prior to the start of re-oxygenation. (D) Bicistronic expression of the indicated His-OTUB1-MBP-FIH in E.coli followed by immunoblot analysis. Time points indicate the time after induction of protein production by addition of isopropyl-ß-D-thiogalactoside (IPTG). Nx, normoxia; M, monomer. Data are representative for three independent experiments throughout.