Nanovue nanodrop

Clearance of UV photoproducts was assessed in whole skin biopsies as previously reported (Jarrett et al., 2014 (link)). Briefly, mice pre-treated (twice a day for 3 days) with vehicle or forskolin were depilated 4 days prior to radiation (7.5 kJ/m²). 1 cm² skin biopsies were collected at the indicated time points after UV exposure, DNA was isolated by DNEasy kit (Qiagen, Germantown, MD) and quantified by Nanovue nanodrop (GE Biosciences, Pittsburgh, PA). 0.1 μg/well DNA was loaded onto nitrocellulose membranes using a slot blot apparatus. Membranes were processed as described (Wang, Wu, & Friedberg, 1991 (link)) and immunoblotted for [6,4]-photoproducts using a monoclonal antibody against this UV photolesion (Cosmo Bio, Tokyo, Japan). Band intensities were analyzed using ImageJ software.

Immuno slot blots were performed on whole cell lysates with 6,4 and CPD antibodies via standard methods^{11 (link)}. Genomic was isolated using the DNEasy Qiagen Kit per manufacturer’s instructions. DNA concentration was determined by Nanovue nanodrop (GE Healthcare) reading. Equal loading of DNA was confirmed by 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, 1mg/ml; Thermo Fisher) staining. DNA was denatured at 95°C for 10 minutes, and 0.1 µg was loaded per well on a slot blot apparatus (BioRad) on a nitrocellulose membrane (BioRad). Wells were washed with TE and suction was applied until dry. Membranes were baked (80°C, 1h), blocked in 5% milk (20°C, 1h, TBS buffer, 0.5% Tween), incubated with anti-CPD (Cosmo Biosciences, 1:1,000 dilution, 4°C, overnight), washed, incubated in secondary antibody (HRP-anti-mouse, Abcam, 1:10,000, 20°C, 1h) prior to visualization by ECL using the STORM system.