Ubiquitin antibody p4d1

E64, leupeptin, aprotinin, and Nonidet P-40 detergent were obtained from Sigma-Aldrich. Z-VVR-AMC substrate was purchased from Enzo Life Sciences. The Z-Arg-Arg-pNA.2 HCl substrate was purchased from Bachem. Mouse monoclonal anti-actin clone C4 antibody was purchased from MP Biomedical, LLC. Antibodies to EhCP4 and the CP inhibitors WRR483 and WRR605 were a kind gift from Dr. Sharon Reed, University of California, San Diego. EhCP5 and EhCox1 like antibodies were generated in rabbits using recombinant proteins expressed in E. coli (Belley and Chadee, 2000 (link)). Ubiquitin antibody (P4D1) was from Cell Signaling Technology and cycloheximide from Sigma-Aldrich.

Synchronized young adult animals were treated with Bortezomib (60 μM) for 8 h and worm pellets were washed 4 times and flash frozen in liquid nitrogen. Polyubiquitinated proteins were enriched using Ubiquitin Enrichment Kit (Thermo Scientific 89899) and eluted with 2× laemmli buffer. Lys48-specific anti-Ubiquitin rabbit clone Apu2 (Sigma 05-1207) was used for immunoprecipitation at 4 °C for 4 h. The immune-complex was captured using Pierce MS-Compatible Magnetic IP Kit Protein A/G (ThermoFisher 90409) and eluted with 2× laemmli buffer. Eluted proteins from both enrichment methods were resolved by SDS-PAGE and gel slices above 50 KDa were sent for protein ID using mass spectrometry. Ubiquitin antibody (P4D1, Cell Signaling Technology, 3936) was used for western blot to detect ubiquitinated proteins at 1:1000 dilution. Mass-spec and protein identification were performed by the UTSW proteomics core. For protein identification, samples were run on a Q-Exactive HF mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Data analysis was performed using Proteome Discoverer 3.0 SP1 using the C. elegans protein database from UniProt.