FIH-1/HIF-1AN Antibody (162c) - BSA Free

Western Blot: FIH-1/HIF-1AN Antibody (162c) - BSA Free [NBP1-30333] -

Hypoxia sensitivity of the FIH-dependent FIH-OTUB1 heterodimer formation. (A) Heterodimer (HD) formation was sensitive to hypoxia (Hx, 0.2% O2), DMOG and DFX, but not to the PHD-specific inhibitor FG-4592. (B) Immunoblotting of cell lysates with the indicated ectopic expressions. The H199A FIH mutant is catalytically inactive and was incapable of forming the HD. (C) Hypoxia sensitivity of HD formation in comparison with HIF-1 alpha and HIF-2 alpha stabilization following 24 h of incubation at the indicated O2 levels. (D) Quantification of the experiment described in (C) and calculation of the oxygen sensitivity of HD formation and HIF-1 alpha and HIF-2 alpha stabilization based on this quantification. Nx, normoxia; M, monomer. Data are shown as mean +/- SEM from four independent experiments or are representative for (A, B) three or (C) four independent experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31299612), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: FIH-1/HIF-1AN Antibody (162c) - BSA Free [NBP1-30333] -

Characterization of the FIH-OTUB1 conjugation. (A) Immunoblot analysis of endogenous FIH IP detected the unexpected protein signal (“X"), which was insensitive to 858 mM beta -mercaptoethanol ( beta ME) and 10 mM DTT. The same antibody was used for the FIH IP and subsequent FIH immunoblotting and the anti-beta -actin antibody was derived from the same species as the anti-FIH antibody, leading to the detection of fragments of the IP antibodies (ab; highlighted by open arrows). Black arrows highlight specific signals of the indicated proteins. (B) The intensity of X detected by immunoblotting of cell lysates was proportional to FIH and wildtype OTUB1 (WT) levels, as seen with ectopic FLAG-OTUB1 expression, knockdown and ectopic mutant OTUB1 (N22A) expression. Protein signal X was insensitive to 100 mM iodoacetamide, the only agent present that disrupts disulfide bonds. (C) Investigation of the resistance of the FIH-OTUB1 HD interaction to a chaotropic agent by urea-PAGE followed by immunoblotting. (D) Investigation of the effect of treatments disrupting thioesters and oxyesters using purified HD. The samples were analysed by Coomassie-stained SDS-PAGE, which included boiling in the presence of 858 mM beta ME during sample preparation. (E) Residues of the FIH interaction site of OTUB1 were mutated and their relevance for HD formation investigated by ectopic expression of the mutated proteins and anti-FLAG-IP. All samples were boiled in the presence of 858 mM beta ME or 10 mM DTT as indicated followed by immunoblotting exp, exposure; HD, heterodimer; M, monomer; ect, ectopic (plasmid-based) expression; end, endogenous protein. The data represent (A) two, (C, D, E (IPs)) three, (B) four or (E (lysates)) six independent experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31299612), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: FIH-1/HIF-1AN Antibody (162c) - BSA Free [NBP1-30333] -

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-beta -D-thiogalactoside (IPTG). Nx, normoxia; M, monomer. Data are representative for three independent experiments throughout. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31299612), licensed under a CC-BY license. Not internally tested by Novus Biologicals.