Tnt kit

Electrophoretic mobility shift assay was performed using ³²P-labeled oligonucleotides and HIF-1α and ARNT proteins prepared by using the TNT kit from Promega. Protein-DNA complexes were resolved by electrophoresis through 5% polyacrylamide gel in 0.5×Tris borate-EDTA at 4°C for 3 h. For oligonucleotide competition experiments, unlabeled oligonucleotides were added to the reaction at 50-fold molar excess to the ³²P-labeled probes. The EMSA probe sequences are listed in the Supplementary Table.

Extract was pre-treated with UbVS (10 μM) for 10 min at 24°C before addition of ubiquitin (50 μM) and substrates. The preincubation time with UbVS was extended to 30 minutes when recombinant DUBs were added to the extract. Pre-treatment of extract with MG262 (Boston Biochem, I-120) (200 μM) or specific DUB inhibitors and their respective control compounds was done at 24°C for 30 minutes. Substrates were expressed and labeled using ³⁵S-methionine (Perkin Elmer, NEG709A500UC) with the TNT kit (Promega: L1770). Each substrate was amplified with primers by PCR (using as templates plasmids from the hORFeome Database) to allow T7-dependent transcription of the PCR product or transcribed directly if plasmids contained a T7 promoter. The translation reaction mix was added to the Xenopus extract at 8% final volume. Samples of the reactions were collected at the indicated time, quenched with SDS sample buffer, and processed for SDS gel electrophoresis and phosphor imaging (Bio-Rad PMI). Quantification was performed using Quantity One software (Bio-Rad).