No relocalization or post-translational modifications of some components of the replication machinery after UVA radiation.
Asynchronous MRC5Vi cells or synchronized in early S-phase (condition S0R, see Material and Methods) are untreated or exposed to 80 and 160 kJ/m2 of UVA radiation. (A) Subcellular localization of Cdc6, Cdc7, Dbf4, Cdc45, Mcm2, Mcm10, Orc2, PCNA, RPA32 in asynchronous cells (Asyncro.) or cells synchronized in early S-phase (Synchro.). (B) The fractions of chromatin-bound proteins and total soluble proteins were recovered at various time points post UVA. The expression levels of Cdc6, Cdc7, Dbf4, Cdc45, Mcm2, p-Mcm2(Ser40/41), Mcm10, Orc2, PCNA, and RPA32 were detected by Western blotting in denaturing and reducing conditions. Lamin A/C and GAPDH were used as loading control for the chromatin-bound and soluble fractions, respectively. The apparent molecular weight of each protein is indicated on the right side of each blot (panel 7A). The stars (*) indicate the position of the non specific bands detected by Orc2 antibody (H-300, SCBT).