Anti phospho rpa32 s4 s8

The methodology was described previously^{29 (link),30 (link)}. The anti-HSC70 antibody was purchased from Santa Cruz (Santa Cruz, CA). Anti-cyclin A2, anti-phospho-ATR (Thr-1981), anti-phospho-Chk1 (Ser-345), anti-phospho-CDK1/2 (Tyr-15), and anti-γ-H2AX (Ser-139) antibodies were from Cell Signaling (Danvers, MA). Anti-phospho-RPA32 (S4/S8) and anti-BRCA2 antibodies were from Bethyl Laboratories (Montgomery, TX). Anti-phospho-histone H1 antibody was from Millipore-Sigma (Burlington, MA). To avoid the cross-reaction of secondary antibodies with previously probed primary antibodies, blots were stripped and re-probed with next primary antibodies raised in a different species in an alternate fashion. All images were acquired and processed using the G: BOX gel documentation system and the GeneSnap software (Syngene, Frederick, MD). Brightness and contrast of images were applied equally across the entire gel/blot image and to controls. Images of protein bands were cropped and only brightness-adjusted using the PowerPoint software (Microsoft, Redmond, WA). Quantification of the protein band intensity was performed using the ImageJ software (NIH, Bethesda, MD).

Cell lysates were prepared by sonicating in UTB buffer (8 M urea, 50 mM Tris HCl [pH 7.4], 150 mM β-mercaptoethanol). Following protein concentration determination using the Bradford reagent (Bio-Rad), lysates were suspended in 2× Laemmli sample buffer (Bio-Rad) and heated to 95°C for 10 min. Lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard procedures.
The following primary antibodies were used for Western blotting: anti-K8α/K-bZIP (SAB5300152; Sigma-Aldrich), anti-ORF6/SSB (provided by Gary Hayward), anti-K8.1A (in-house), anti-β-actin (A2228; Sigma-Aldrich), anti-ATM (2873; Cell Signaling), anti-phospho-ATM (S1981) (AF1655; R&D Systems), anti-CHK2 (2662; Cell Signaling), anti-phospho-CHK2 (T68) (2661; Cell Signaling), anti-RPA32 (NA19L; Calbiochem), anti-phospho-RPA32 (S4/S8) (A300-245A; Bethyl), anti-H2AX (7631; Cell Signaling), anti-γH2AX (S139) (05-636; Merck Millipore), anti-MRE11 (GTX70212; Genetex), anti-NBS1 (GTX70222; Genetex), anti-RAD50 and anti-KU80 (sc-6954; Santa Cruz), and anti-KU70 (ab83501; Abcam). Goat anti-mouse and swine anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako Laboratories) and ECL detection reagent (GE Healthcare) were used to visualize proteins on a Fusion SL chemiluminescence imaging system (Vilber Lourmat).

The following antibodies were used in this study: anti-FLAG (F3165, Sigma, St Louis, MO, USA); anti-BRCA1 (D-9, Santa Cruz, Dallas, TX, USA); anti-cyclin A (sc-751 and sc-271645, Santa Cruz); anti-NBS1 (A301-289A, Bethyl, Montgomery, TX, USA); anti-53BP1(612522, BD, San Jose, CA, USA); anti-phospho-ATM (S1981) (4526, Cell Signaling, Danvers, MA, USA); anti-phospho-RPA32 (S4/S8) (A300-245, Bethyl); anti-phospho-53BP1 (S25/29) (2674, Cell Signaling); anti-phospho-53BP1 (S1778) (2675, Cell Signaling); anti-phospho-NBS1 (S343) (3001, Cell Signaling); anti-phospho-SMC1 (S957) (4801, Cell Signaling) anti-phospho-CHK1 (S345) (2348, Cell Signaling); anti-phospho-KAP-1 (S824) (4127, Cell Signaling) and anti-phospho-(Ser/Thr) ATM/ATR substrate antibody (2851, Cell Signaling). Rabbit anti-PTIP antisera were obtained from rabbits immunized with GST-PTIP (residues 590–1 069) fusion protein expressed and purified from Escherichia coli. Antisera were affinity-purified using AminoLink Plus Immobilization and Purification Kit (Pierce, Waltham, MA, USA ). Rabbit polyclonal antibodies against BRCA1, RIF1, ATM, γH2AX, 53BP1 and phospho-CHK2 (T68) were described previously [5 (link), 31–33 ].