Mab375

The recombinant extracellular domains of the receptors DR5 (100 ng/well), VEGFR1 or VEGFR2 (50 ng/well) (R&D Systems, Minneapolis, MN, USA) were immobilized on ELISA plates overnight at 4 °C in 0.1 M carbonate–bicarbonate buffer (pH 9.4). The plates were washed three times with PBST (phosphate-buffered saline + 0.05% Tween), and wells were blocked by 2% BSA in PBST for 1 h at 37 °C. After blocking, dilutions of DR5-B, HRH–DR5-B or SRH–DR5-B (in 3 replicates) at concentrations from 0.032 to 2500 nM were added, and the plates were incubated for 1 h at 37 °C. Captured ligands were detected by subsequent incubation with monoclonal antibodies to TRAIL (MAB375, R&D Systems, Minneapolis, MN, USA) and anti-mouse polyclonal goat IgG conjugated with horseradish peroxidase (HAF007, R&D Systems). Unbound antibodies were washed 3 times with a PBST buffer, and color was developed by OPD (o-phenylenediamine dihydrochloride) colorimetric substrate. After 15 min of incubation with the substrate at 37 °C, the reaction was stopped by a 1 N H₂SO₄ solution. The optical density was determined at 450 nm by iMark reader (Bio-Rad, Hercules, CA, USA). Dissociation constant (K_D) values were calculated by GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA), using the nonlinear regression option in the XY analysis section.

Mouse dorsal fur was removed via shaving and depilation with Veet (Reckitt Benckiser, UK). Mice were placed in restrainers with facial protection and treated once ±250mJ/cm² UVB light using the UV-2 ultraviolet irradiation system (Tyler Research). UVB light was provided by cascade-phosphor ultraviolet generators that emit 310nm UVB radiation.
For irradiation of cultured cells at approximately 80% confluence, growth medium was replaced with PBS and cells were treated with 0-50mJ/cm² UVB light as indicated in specific experiments. PBS was removed immediately following irradiation and replaced with growth medium. Cells were incubated at 37°C for the indicated times.
Inhibitors of cell death (Ac-YVAD-cmk, Sigma Aldrich SML0429; Necrostatin-1, Cayman Chemical 11658; GSK’872, Tocris Bioscience 64-921-0; Z-IETD-FMK, Selleckchem S7314; Z-LEHD-FMK, Selleckchem S7313; Z-VAD-FMK, Selleckchem S7023; UAMC-3203, Selleckchem S8972) were added 30 minutes prior to IFN treatment and again immediately following UVB exposure.
For neutralizing antibody experiments, N/TERTs were treated with isotype control (R&D MAB002, MAB0041) or neutralizing antibodies targeting TRAIL (100ng/ml; R&D MAB375), TNF-α (5µg/ml; R&D MAB210) or FasL (1µg/ml; R&D MAB126) immediately following UVB exposure.