Anti rad51

Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-RB (GeneTex, Irvine, CA, USA), anti-phospho-Akt(Ser473) (Cell Signaling), anti-Akt (Cell Signaling), anti-phospho-Erk1/2(Thr202/Tyr204) (Cell Signaling), anti-Erk1/2 (Cell Signaling), anti-TOP2A (Cell Signaling), anti-CCND1 (Cell Signaling), anti-γH2AX (Cell Signaling), anti-CDK2 (GeneTex), anti-RAD51 (GeneTex), anti-CDK4 (Cell Signaling), and anti-GAPDH (GeneTex) antibodies followed by horseradish peroxidase-conjugated secondary antibodies and visualization using an enhanced chemiluminescence detection system. Immunohistochemical (IHC) sample preparation and staining with anti-GFAP (Genetex), anti-phospho-RB (Cell Signaling), anti-Ki-67 (Cell Signaling), and anti-γH2AX (Cell Signaling) antibodies were carried out as previously described [48 (link)]. Uncropped Western blot images are provided in Figure S5.

Exponentiall growing cells were collected by trypsinization, washed with PBS, and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 1% Sodium deoxycholate and 0.1% Sodium dodecylsulfate) containing protease inhibitor cocktail (Roche Japan, Tokyo, Japan). Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific), and 8 µg of proteins were electrophoresed on 5–10% SDS–polyacrylamide gels, and then electrophoretically transferred to a polyvinyl difluoride membrane. Primary antibodies used in this study are as follows: anti-RAD51 (GTX70230, GeneTex), anti-MRE11 (clone 12D7, GeneTex), anti-ATM (clone 1A1, GeneTex), anti-Rad50 (clone 13B3, GeneTex), anti-NBS1 (clone 1D7, GeneTex), anti-BRCA1 (clone 6B4, GeneTex), anti-BRCA2 (clone 1B8, GeneTex), anti-Ku86 (clone 111, Kamiya Biochemical co., Seattle, WA, USA), anti-Ku70 (clone N3H10, Kamiya Biochemical co.), anti-DNA-PKcs (MC-365, Kamiya Biochemical co.), anti-XRCC4 (GTX70293, GeneTex), anti-Lig IV (GTX74360, GeneTex), anti-XLF (A300-730, BETHYL Laboratories, Inc.), and anti-γ-tubulin (clone DTU-88, Sigma-Aldrich).

Primary antibodies used in this study were as follows (Table S1): Anti-RecQL4 antibody was prepared by Abfrontier by immunizing rabbits with recombinant N-terminal (amino acid residues 1–241) RecQL4. Other antibodies used in this study were as follows: Anti-pATM (Ser-1981, #4526, Cell Signaling), anti-Mre11 (GTX30294, GeneTex), anti-Nbs1 (A7703, ABclonal), anti-Rad50 (sc-74460, Santa Cruz), anti-Rad51 (GTX100469, GeneTex), anti-RPA32 (MABE-286, EMD Millipore), anti-Usp28 (A9292, ABclonal), anti-Skp2 (sc-7164, Santa Cruz), anti-γH2AX (A300-081A, Bethyl and 05-636, EMD Millipore), anti-lamin B1 (ab16048, Abcam), anti-GAPDH (sc25778, Santa Cruz), anti-FLAG M2 (Sigma), and anti-HA (AE008, ABclonal). The following antibodies were used as secondary antibodies in immunofluorescence microscopy: Alexa Fluor 488 anti-mouse IgG (A11001, Thermo Fisher), Alexa Fluor 488 anti-rabbit IgG (A11008, Thermo Fisher), Alexa Fluor 594 anti-mouse IgG (A11005, Thermo Fisher), and Alexa Fluor 594 anti-rabbit IgG (A11012, Thermo Fisher).