Anti uba6 antibody

ORF of human UBA6 and USE1 in pEnter, with C terminal Flag, were purchased from Vigene Biosciences (JiNan, China). Moreover, UBA6 with the UFD domain (residues 949-1052)deletion (UBA6^ΔUFD) was generated by PCR. The amplified DNA fragment was cloned into pEnter. The human embryonic kidney cell line HEK293 was purchased from ATCC and was cultured in DMEM supplemented with 10% of FBS and 50 U/ml of penicillin/streptomycin. Cells were transfected using Lipofectamine 2000 reagent as described by the manufacturer’s instructions. 500 μM of inosine or vehicle was added 24 h after transfection. At 48 h, cells were harvested and lysed in lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TRITON X-100). Cleared lysates were subjected to anti-FLAG immunoprecipitation using Anti-FLAGM2 Affinity Gel (Sigma, USA) overnight at 4 °C. Samples were washed three times with TBS. Proteins were separated on 8% or 12% Laemmli SDS gels and subjected to western blot analysis using an anti-UBA6 antibody (Proteintech, 1:1000), anti-USE1 antibody (ABclonal, 1:1000), and anti-β-Actin antibody (Sangon Biotech, 1:1000).

Standard immunohistochemical (IHC) assays were performed for UBA6 evaluation as described previously^{66 (link)}. In brief, tumours were harvested before immunotherapy and fixed in 10% neutral-buffered formalin. After deparaffinization and rehydration, 4 μm tissue sections were subjected to heat-induced epitope retrieval. Slides were processed with the VECTASTAIN Elite ABC HRP Kit and DAB Substrate Kit (Vector Laboratories). Slides were then incubated with anti-UBA6 antibody (Proteintech, 1:1500). Five visual fields from different areas of each slide were independently evaluated by two pathologists who were blinded to the group allocation during the staining and when assessing the outcomes. Necrotic areas in the tumours were excluded from the evaluation. IHC intensity scores of UBA6 were ranked into four groups: negative (−), positive-low (+), positive-medium (++), and positive-high (+++). In the IHC scoring of patient samples, the score “low” corresponded from negative (−) to positive-low (+), while the score “high” corresponded to the range from ++ to +++. To stain for CD8, slices were incubated with an anti-CD8 antibody (Cell signalling #85336 S, 1:200). CD8-positive cells were analysed using the Image J cell counter. The average infiltration of CD8⁺ cells and average expression of UAB6 in the tumour tissues were assessed.

Recombinant human ubiquitin and FAT10 were purchased from Boston Biochem. Plasmids pEnter-UBA6 and pEnter-USE1 were used for the expression of Flag-UBA6 and Flag-USE1. The two plasmids were transfected into HEK293, respectively, using Lipofectamine 2000 reagent. Purification of Flag-UBA6 and Flag-USE1 was carried out using Anti-FLAGM2 Affinity Gel (Sigma) as described by the manufacturer’s instructions. Flag-UBA6 (0.5 μM) and Flag-USE1 (0.5 μM) were incubated with vehicle, inosine, or TAK243 at room temperature for 15 min. Then, ubiquitin (5 μM) or FAT10 (2 μM) with ATP (250 μM) were added. The reaction mixtures were incubated at 37 °C for 30 min before 2× Lammli sample loading buffer was added to quench the reaction. The thioester detection was fractionated by SDS-PAGE under nonreducing conditions and immunoblotted with anti-UBA6 antibody (Proteintech, 1:1000) and anti-USE1 antibody (ABclonal, 1:1000).