Anti v5 mab

For DTX3LM validation, HEK293T cells were transfected with FLAG-PARP9-c-Myc–DTX3L or FLAG-PARP9M-c-Myc–DTX3LM expression vectors for 30 h, incubated with 20 μM MG-132 (Enzo Life Sciences) for 14 h, and then subjected to lysis and Immunoblotting with c-Myc Ab. For EMCV-3C degradation, EMCV-3C cDNA was amplified from EMCV virus stock, confirmed with sequencing, tagged with V5, and inserted into the pcDNA3.1 expression vector. The pCI-His-Ub plasmid was a gift of A. Winoto (Addgene plasmid #31815), and pHA-Ubiquitin plasmid was a gift of E. Yeh (Addgene plasmid #18712). The TRIM22 plasmid was from GeneCopoeia. Cells were transfected with V5-EMCV-3C and His-Ub vectors using X-tremeGene 9 (Roche), and 36 h later were treated with or without 20 μM MG-132 for 14 h before lysis and Immunoblotting. For EMCV-3C ubiquitination, cell lysates were immunoprecipitated with anti-V5 mAb conjugated to agarose (Sigma), eluted with 1% SDS, subsequently diluted to 0.1% SDS, reprecipitated with anti-V5 mAb (Invitrogen), and then Immunoblotted with anti-HA Ub mAb (Sigma) followed by anti-V5 Ab.

The mAbs were initially mapped against MDTCS and T2-CUB2. Binding to ADAMTS13 was used as a reference. Purified mAbs were coated individually on a 96-well microtiter plate. After blocking (3% milk in PBS), plates were incubated with a serial dilution of MDTCS, T2-CUB2 or ADAMTS13, starting at 15 nM (1 h at 37 °C). Binding was detected with the HRP-labeled anti-V5 mAb (Invitrogen, 1/3000 in PBS, 0.3% milk). Colorimetric development was done using OPD and H₂O₂ and was stopped using 4 M sulfuric acid, after which absorbance (490 nm) was determined. Epitope mapping of anti-MDTCS mAbs was refined using MDTCS variants (Fig. 1B) as described above, except that expression medium was used and that binding was detected with the biotinylated mAb 3H9 (3H9bio). Secondary detection was performed with HRP-conjugated streptavidin (1/10,000 in PBS, 0.3% milk). Epitopes of the anti-T2-CUB2 mAbs were mapped using the T2-CUB2 variants (Fig. 1C and D). Detection was done with anti-V5-HRP. Competition of the anti-T2-CUB2 mAbs directed against the same domain was tested by adding biotinylated mAbs (EC₅₀), together with a dilution of the respective non-biotinylated mAb, to ADAMTS13 (8.6 nM), captured with 3H9 (5 µg/mL). Binding was detected with HRP-conjugated streptavidin.