Western Blot Analysis of Protein Expressions

Proteins were isolated and their differential expressions were analyzed by Western blot, as described previously [21 (link),22 (link)]. Briefly, the cells were lysed using RIPA buffer (50 mM Tris HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100 or NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 10 mM NaF freshly supplemented with protease and phosphatase inhibitors). Protein concentrations were quantified using a Bradford assay (Bio-Rad), and the samples were normalized for equal loading. The samples were then heated to 100 °C for 10 min in the presence of 6× Laemmli buffer (Boston BioProducts; Milford, MA, USA). The proteins were resolved by SDS-PAGE and their expression was analyzed following immunoblotting using specific antibodies. The following antibodies were used in this study: FANCD2 (R&D MAB93691), pCHK2-T68 (Invitrogen PA5-104715), pRPA 32-S33 (Cell Signaling 10148s), pCHK1-S296 (Cell Signaling 90178s), pH2AX-S139 (Millipore 05-636), GAPDH (Santa Cruz 32233), and Vinculin (Cell Signaling 13901S).