Pr 619

The NRK-49F cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan) containing 5% fetal bovine serum (FBS) (Nichirei Bio Science, Tokyo, Japan) and penicillin/streptomycin (Nacalai Tesque). To test the effects of PR-619 on the cultured cell line, 3 μmol/L PR-619 (LifeSensors) was applied to subconfluent cells for 60 min before TGF-β1 stimulation. Cells were then exposed to TGF-β1 (10 ng/mL) in the presence or absence of PR-619 for 24 h. Whole cell lysates were prepared and subjected to western blot analysis.

Pulldown of polyubiquitin protein conjugates was performed using agarose Tandem ubiquitin-binding entities, TUBEs (Lifesensors, UM402) according to the manufacturer’s protocol. Both G2-arrested cells with 5 µM etoposide for 18 h and asynchronously growing cells were treated prior to cellular lysis with 40 µM MG-132 for 1 h. Cells were lysed for 1 h at 4 °C in TUBE lysis buffer (Tris-HCl 50 mM pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1% NP40, 10% Glycerol supplemented with phenanthroline 100x and 50 µM PR-619 from Lifesensors) and protein lysates (~0.5 mg) were incubated with agarose TUBEs for 3 h at 4 °C. Bound proteins were resolved in 7.5% SDS–PAGE in parallel with the unbound fraction for subsequent immunoblot analysis.

Mice of appropriate age and genotype were asphyxiated with CO₂ or deeply anesthetized via isoflurane prior to rapid decapitation. Tissues were removed and homogenized in a modified RIPA buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5 mM EGTA, 1 mM EDTA, 0.5% SDS, 1% Triton X-100, and 1% sodium deoxycholate. Complete protease inhibitors (Roche, Indianapolis, IN), phosphatase inhibitor cocktail I (Sigma Aldrich, St. Louis, MO), and 50 μM PR-619 (Life Sensors, Malvern, PA) were added to the homogenization buffer. Samples were homogenized in 1X Laemmli buffer, sonicated, and boiled. After homogenization, tissues were centrifuged at 17,000 x g for 10 min at 4°C, and supernatants were removed and immediately frozen at—80°C. Protein concentrations were determined by using the bicinchoninic acid (BCA) protein assay kit from Pierce (Rockford, IL).

To identify Cul4b and DCAF1-interacting proteins in T cells, CD4⁺ T cells were purified by MACS and activated with anti-CD3/CD28 mAbs for 40 h. Cells were lysed in Triton-X lysis buffer (1% Triton-X, 50 mM Tris (pH 8), 100 mM NaCl, containing EDTA-free complete protease inhibitor cocktail (Roche), deubiquitylase inhibitors PR619 (LifeSensors), and Zn2+-chelator o-PA). The lysates were cleared by centrifugation at 15,000 rpm at 4°C for 12 min. The supernatant was divided into 3 parts and incubated with Dynabeads (Invitrogen) conjugated either with Cul4b antibody or DCAF1 antibody or rabbit IgG in cold room overnight. The immunocomplex was washed 4 times with PBS-Tween (PBST, 0.2% Tween), and each sample was eluted from beads with 0.3% SDS and acetone/TCA precipitated. The resulting pellet was digested with the iST kit (PreOmics GmbH, Martinsried, Germany) per manufacturer’s protocol [88 (link)]. Briefly, solubilization, reduction, and alkylation was performed in sodium deoxycholate (SDC) buffer containing TCEP and 2-chloroacetamide. Samples were enzymatically hydrolyzed for 1.5 h at 37°C by LysC and trypsin. To stop the digestion, the reaction mixture was acidified with 1% TFA in isopropanol. Peptides were desalted and eluted and dried by vacuum centrifugation, then reconstituted in 0.1% TFA containing iRT peptides (Biognosys).

HEK293T cells were transfected with a PiggyBac transposon-based plasmid (a kind gift from Dr. Pentao Liu [57 ]) for Tax-S-Tag. Cells were harvested 48 hours later and lysed with lysis buffer containing a deubiquitinase inhibitor, PR619 (Life Sensors). Cleared cell lysate was then incubated with S-protein agarose beads (Novagen) at 4^°C overnight. The beads were washed three times with lysis buffer and the protein was then eluted in equal volume of 2X Laemmli Sample Buffer (Sigma-Aldrich). The eluted protein was heated to 95^°C for 10 minutes for immunoblotting.