Xpress tag

IVTT reaction mixtures (100 μl) were incubated at 4°C for 1 h with a 50% slurry of PBS/Glutathione‐Sepharose 4B (GE Healthcare) with gentle mixing. Glutathione‐Sepharose was pelleted in Pierce spin cups (Thermo Fisher) by centrifugation at 1000 × g for 1 min and washed 8 times in Tris‐buffered saline (pH 7.2) containing 1% Triton X‐100. Proteins bound to the Glutathione‐Sepharose beads were eluted using a 10 mM glutathione buffer (50 mM Tris, 10 mM reduced glutathione, pH 8.0). The final eluted protein mixes were then used in EMSA's (see next section) or processed for SDS‐PAGE analysis by resuspending in SDS‐PAGE loading buffer and boiling for 10 min. Proteins were separated by SDS‐PAGE on a 4–20% gradient gel and then immunoblotted as described above using antibodies directed against the GST tag (EMD Millipore) to detect AntitoxP, V5 tag to detect ToxP or the Xpress tag (Invitrogen) to detect CBU0665.

Full-length human ITCH cDNA was amplified from human Hela cDNA and cloned into the pcDNA3.1 (Xpress-tag, Invitrogen) and pET-32a(+) (Novagen) vectors. Ala substitution at S257 of ITCH and Arg substitution at K46 of H1.2 was performed using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). WT ITCH, S257A ITCH, WT H1.2, and K46R H1.2 cDNAs were cloned into pEGFP-C1 (Clontech), a gift from Dr. Yanzhong Yang (City of Hope). ITCH deletion mutants were generated by PCR and subcloned into Xpress-tag pcDNA3.1. ITCH shRNA and scrambled control shRNA were purchased from MISSION shRNA at Sigma-Aldrich. HEK293T or MDA-MB-231 cells were transfected using PolyJet DNA transfection Reagent (SignaGen). DDK-tagged H1.2, H1.3 and H1.4 plasmids were purchased from OriGene Technologies, Inc. pRK5-HA-ubiquitin-K63, pRK5-HA-ubiquitin-K48, and pRK5-HA-ubiquitin-K29 were purchased from Addgene.

Cell imaging was performed in 384-well plates. For living cells, 30 min prior imaging, 25 μl of culture medium were removed and replaced by 25 μl of 2× mix of dyes, to a final concentration of 200 ng/ml of Hoechst H33342 (nuclear staining; Life Technologies), 10 nM of TMRM (mitochondrial membrane potential; Life Technologies), and/or 1/100 Annexin-V-Alexa Fluor 647 (early apoptosis; Life Technologies). If chemical inhibitors of the ETC were used in the experiments, they were added to hMDMs at the indicated times points at the following concentrations: 5 μM oligomycin (Enzo), 100 μM DCCD (Sigma), 10 μM FCCP (Tocris), and 50 μM BTB06584 (Sigma). Once the assay was performed, cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100, blocked with 1% BSA, and stained with primary mouse antibodies against Xpress tag (1:100, Invitrogen) and secondary anti-mouse Alexa Fluor 488 antibodies (1:1000, Invitrogen). Image acquisitions of multiple fields (9–25) per well were performed on an automated confocal microscope (Opera Phenix, PerkinElmer) using ×40 or ×60 objective, excitation lasers at 405, 488, 561, and 640 nm, and emission filters at 450, 540, 600, and 690 nm, respectively.