Na935

Immunoblotting was performed as previously described (16 (link), 17 (link)), except that cell lysates or membrane fractions were analyzed using nonreducing (-DTT) SDS-PAGE for detecting CD55. Blotted polyvinylidene fluoride membranes (Millipore) were blocked for 1 h with 5% skim milk and 0.1% Tween 20 in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mM NaH₂PO₄, pH 7.4) and incubated with primary antibodies (Table S4) diluted in blocking solution. The blot was washed in 0.1% Tween 20 in PBS, followed by incubation for 1 h with 1:3000 dilutions of horseradish peroxidase–conjugated antimouse, anti-rabbit, or antirat secondary antibodies (NA931, NA934, or NA935; Cytiva). Immunoreactive bands were detected using Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore) or SuperSignal West Femto maximum sensitivity substrate (Pierce; Thermo Fisher Scientific) according to the manufacturer’s instructions. Chemiluminescence images were obtained using ImageQuant LAS 500 (Cytiva).

DNA was extracted using PureLink Genomic DNA Mini Kit (Invitrogen, K1820-01). DNA (200–500 ng per well, two technical replicates per sample) was vacuum-transferred to a nitrocellulose membrane using the Bio-Dot or bio-dot SF apparatus (Bio-Rad, 1706542/5, Rishon LeZion, Israel). The membrane was baked at 80 °C for 90 min in a Bio-Rad’s Gel Dryer model 583, blocked in 5% milk, then incubated with damage-specific primary antibodies: anti 6-4 PP diluted 1:2000 (Cosmo bio, NM-DND-002, Otaru, Hokkaido, Japan), anti CPD diluted 1:4000 (Cosmo bio, CAC-NM-DND-001, Otaru, Hokkaido, Japan), and anti-cisplatin diluted 1:5000–1:15,000 (abcam, 103261, Cambridge, UK). After incubation with an HRP-conjugated secondary antibody, NA931 or NA935 (Cytiva, Marlborough, MA, USA) for UV damage and cisplatin damage, respectively, the damage signal was detected using Enhanced chemiluminescence (ECL™ Prime Western Blotting System, Cytiva, RPN2232, Marlborough, MA, USA). Genomic DNA amount loaded onto the membrane was quantified using SYBR™ Gold Nucleic Acid Gel Stain (Invitrogen, S11494, Carlsbad, CA, USA), and the damage signal was normalized according to SYBR-Gold signal using ImageJ software version 1.5.1j8 and Image Lab version 6.0 Bio-Rad.