Immunoblotting was performed as described12 (link) with the following Abs: 53BP1 (175933, Abcam; NB100-304, Novus Biological); ATR (sc-1887, Santa Cruz Biotechnology); BRCA1 (MAB22101, R+D systems); Chk1 (sc-8408, Santa Cruz Biotechnology); Chk1-S345-P (#2341S; Cell Signaling Technology); Chk2 (BD 611570, BD Biosciences); flag-tag (M2, Sigma; F1804, Sigma); γtubulin (GTU488, Sigma); MAD2L2/Rev7 (ab180579, Abcam); myc-tag (9B11, Cell Signaling Technology); OBFC1/Stn1 (E10-376450, Santa Cruz Biotechnology); Tagged Ten1 was not detectable by immunoblotting of transfected 293T cells.
For detection of RPA phosphorylation, conditional CTC1 HCT116 cells or MEFs were irradiated and harvested 3 h later. Cells were washed in PBS, and then collected by scraping in Laemmli sample buffer, boiling for 5 min, and shearing through a syringe. Proteins were separated by SDS-PAGE on 8-16% Tris-Glycine gradient gels (Invitrogen), and transferred to nitrocellulose overnight. Immunoblotting for pRPA followed standard protocols with blocking in 5% milk/TBST and the pRPA Ab (S4/S8; Bethyl) diluted 1:1000 in 1% milk/TBST.
For detection of RPA phosphorylation, conditional CTC1 HCT116 cells or MEFs were irradiated and harvested 3 h later. Cells were washed in PBS, and then collected by scraping in Laemmli sample buffer, boiling for 5 min, and shearing through a syringe. Proteins were separated by SDS-PAGE on 8-16% Tris-Glycine gradient gels (Invitrogen), and transferred to nitrocellulose overnight. Immunoblotting for pRPA followed standard protocols with blocking in 5% milk/TBST and the pRPA Ab (S4/S8; Bethyl) diluted 1:1000 in 1% milk/TBST.