Generating Expression Constructs of HIF1α and Associated Proteins

To make vectors expressing full-length or different fragments of HIF1α with Flag, myc, GFP, or glutathione S-transferase (GST) tags, PCR amplification was performed using pfu polymerase (Yeasen) and the amplicons were inserted into p3xflag-CMV-13, pCDNA3-myc, pEGFP-C1, or pEBG. The constructs harboring EGFP-tagged HOIP and mCherry-tagged HIF1α were generated by cloning the DNA fragments encoding HOIP-EGFP and HIF1α-mCherry into p3xflag-CMV-13. Lamin B1 cDNA was subcloned into pTagBFP-C1 to construct plasmid expressing Lamin B1-BFP. The lentivirus expressing HOIP was engineered by cloning HOIP cDNA into pLvx-t2a-mCherry. The constructs harboring HA-tagged Otulin, CYLD or A20 were obtained by subcloning the PCR products into pCDNA3.1. The plasmid expressing GST-specific tandem Ub-binding entity (TUBE) was constructed by subcloning the cDNA encoding UBAN domain of IκB kinase (IKK) γ into pGEX-4T-1. Site-directed mutagenesis of HIF1α mutants was conducted to generate different point mutants using site-directed mutagenesis kit (Yeasen) as described previously [31 (link), 32 (link)]. Other plasmids were described in the previous publications [6 (link), 31 (link)–39 (link)]. All constructs were verified by DNA sequencing.