Anti cpd

Exponentially growing epimastigotes were pulsed with 100 μM of EdU (Click-iT Edu Image Kit, Invitrogen) for 5 minutes for replication assays or treated with 20 mM of hydroxyurea or UV radiation for DNA damage assays. The cells were pelleted, washed with PBS and fixed with 4% (v/v) paraformaldehyde in PBS for 20 min at room temperature. The cells were permeabilized with 0.1% Triton X 100 for 5 min and washed three times with PBS. After, the cells were incubated with anti-LmRPA-1 [19 (link)], anti-TcRPA-2 or anti-CPD (Cosmo Bio) primary rabbit antibodies for 1 h. Before incubation with anti-CPD, the cells were treated with 2 M HCl for 10 min at 37°C. The coverslips were rinsed three times with PBS and incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 555 for 1 h. After washing the coverslips three times in PBS, the slides were mounted with VectaShield containing DAPI (Vector). For the EdU assay, after permeabilization, the cells were first incubated with a Click-iT reaction cocktail for 30 min at room temperature. Images were acquired through a z-series of 0.2 μm using a 100X 1.35NA lens and Cell R software in an Olympus IX81 microscope. Images were deconvolved using Autoquant X2.1.

The primary antibodies used were as follows (company, catalogue number): anti-ERCC6 (Abcam, ab96098), anti-NANOG (Abcam, ab21624), anti-SOX2 (Santa Cruz, sc-17320), anti-OCT4 (Santa Cruz, sc-5279), anti-SMA (Sigma, A5228), anti-TUJ1 (Sigma, T2200), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-CD90-FITC (BD Bioscience, 555595), anti-CD73-PE (BD Bioscience, 550257), anti-CD105-APC (BD Bioscience, 17-1057-42), anti-IgG-FITC (BD Biosciences, 555748), anti-IgG-PE (BD Biosciences, 555749), anti-IgG-APC (BD Biosciences, 555751), anti-Lamin B (Santa Cruz, sc-6217), anti-LAP2 (BD Bioscience, 611000), anti-Ki67 (ZSGB-BIO, ZM0166), anti-P16 (BD Bioscience, 550834), anti-γ-H2AX (Millipore, 05-636), anti-Nestin (Millipore, MAB5326), anti-PAX6 (Covance, PRB-278P), anti-CPD (Cosmo Bio, TMD-2), anti-cleaved PARP (Cell Signaling Technology, 9541), anti-β-Actin (Santa Cruz, sc69879), anti-GAPDH (Santa Cruz, sc-25778), and anti-hCD31 (BD Bioscience, 555445).

For immunodot blot analysis, samples of 1 µg of DNA per dot were loaded on PVDF membranes (Milipore, Germany). After transfer, membranes were washed with TBS-T (TBS-0.05% Tween 20). After blocking with 5% skim milk, blots were incubated with anti-CPD (Cosmo Bio, Japan) in TBS-T for 12 h followed by washing for twice with TBS-T. Anti-mouse secondary antibody conjugated horseradish peroxidase was incubated for 1 h. After washing, the blots were treated with ECL plus (ELPIS, Korea), and chemiluminescence was detected by X-ray films. The experiments were performed three times independently.