Ubch5

Full-length GST-S100A6 and HA-MDM2 and its fragments were expressed and purified as previously described in the GST pull-down assay. The in vitro ubiquitination assay was performed in a total reaction volume (approximately 100μL) consisting of 5 μg E1 (0.5 μg/μL), 5 μg UbcH5 (1 μg/μL), 50 μg HA-MDM2, 10 μg ubiquitin (1 μg/μL), 50 μg GST-S100A6, and 5 μg 10 × Buffer (1 μg/μL). E1, UbcH5, ubiquitin, and 10 × Buffer were purchased from Enzo Life Science. The in vitro reactions were carried out at 37 °C and 380 rpm for 60–90 min, followed by a western blot assay.

Immortalized human bronchial epithelial Beas-2B cells were purchased from the ATCC and maintained in HITES medium supplemented with 10% FBS and transfected by Nucleofection with Lonza products; HeLa cells were maintained in Eagle's medium with 10% FBS and transiently transfected with Turbofect (Thermo). Cells were maintained in a 37°C incubator with 5% CO₂ as described previously. Anti-IL-22R antibody was from Millipore. Skp-1, phospho-STAT3, STAT3, and phospho-ERK antibodies were from Cell Signaling. V5 antibody, mammalian expression plasmid pcDNA3.1/HisV5-topo, and Escherichia coli Top10 competent cells were purchased from Life Technologies. Immobilized protein A/G beads were obtained from Pierce. Phospho-JNK and JNK antibodies are from Abcam. Cycloheximide, buffering chemicals, and phosphatase inhibitor mixtures were from Sigma. Gel extraction kits and QIAprep spin miniprep kits were from Qiagen. In vitro transcription and translation (TnT) kits were from Promega. DTT, ATP, ubiquitin activating enzyme, UbcH5, UbcH7, ubiquitin, and ubiquitin aldehyde were from Enzo Life Sciences (Farmingdale, NY). Skp-1, Rbx1, and Cul-1 were from Abnova (Taipei, Taiwan), and TnT Coupled Reticulocyte was from Promega (Madison, WI). Talon metal affinity resin was from Clontech (Mountain View, CA). All materials in the experiments are of the highest grade and commercially available.