Mouse anti brdu idu

Primary antibodies used in this study were rabbit anti-GFP (Invitrogen, A-11122, 1/750), chicken anti-GFP (Invitrogen, A-10262, 1/1000), rabbit α-DsRed (Clontech, 632496, 1/750), mouse anti-BrdU/IdU (Becton Dickinson, 347580, 1/25), rabbit anti-PKCα (Santa Cruz, sc-208 1/400), mouse anti-PCNA (Santa Cruz, sc-56, 1/400), rabbit anti-phospho-histone 3 (Upstate, 06-570, 1/500), mouse anti-HuC (Invitrogen, A-21271, 1/200), mouse anti-GS (BD Biosciences, 610517, 1/500), mouse anti-parvalbumin (Chemicon, MAB1572, 1/400) (Inoue and Wittbrodt, 2011 (link)). Secondary antibodies were Alexa488 anti-rabbit and Alexa546 anti-mouse (Invitrogen, A-11034 and A-11030 respectively, 1/400), and DyLight488 anti-chicken, DyLight549 anti-rabbit and Cy5 anti-mouse (Jackson, 703-485-155, 112-505-144 and 715-175-151, respectively, 1/400).

mESCs were treated 40–48 h after passage. mESCs were incubated in culture with 250 µM 5-chloro-2′-deoxyuridine (CldU, Sigma, C6891) for 20 min, washed twice with PBS, incubated with 2 mM hydroxyurea (HU, Sigma, H8627) for 3 h, washed twice with PBS, and incubated with 250 µM 5-Iodo-2′-deoxyuridine (IdU, Sigma I7125) for 20 min. Then, 100 µM IAA (Sigma, I5148), 50 µM mirin (Cayman, 13208), 10 µM PFM01 (Tocris, 622210, Bristol, UK), 10 µM DNA2-IN-C5 (Aobious, AOB9082, Gloucester, MA, USA), 2.5 µM CSN5i-3 (Novartis Pharma, Basel, Switzerland), 25 µM compound 33-11 (ChemBridge Corporation, 6655693, San Diego, CA, USA), or 5 µM ML216 (Aobious, AOB1300) were added at the indicated time points. In place of HU treatment, aphidicolin (Cayman Chemical, 14007) was used at 15 µM. Labeled mESCs were treated with TrypLE and resuspended in PBS at 2 × 10⁵ cells/mL. DNA fiber spreading and immunostaining were performed as previously described [137 (link)]. Primary antibodies used were rat anti-BrdU (CldU) (Abcam, Waltham, USA) and mouse anti-BrdU (IdU) (Becton Dickinson, Franklin Lakes, NJ, USA). Secondary antibodies used were Alexa Fluor anti-rat 568 and Alexa Fluor anti-mouse 488. Antibody information is presented in Supplementary Table S1.

For DNA fiber assay, mESCs were treated with CHEK2 inhibitor, CHEK2 inhibitor and IAA, p53 inhibitor, or p53 inhibitor and IAA 16–18 hr after passaging. After 12 hr of treatment, mESCs were incubated in culture with 30 µM CldU (Sigma) for 20 min, washed twice with PBS, and incubated with 250 µM iododeoxyuridine (IdU) (Sigma) for 20 min. Labeled mESCs were resuspended in PBS at 2 × 10⁵ cells/ml. DNA fiber spreading and immunostaining were performed as previously described (Huang et al., 2013 (link)). Primary antibodies used were rat anti-BrdU (CldU) (Abcam) and mouse anti-BrdU (IdU) (Becton Dickinson). Secondary antibodies used were Alexa Fluor anti-rat 568 and Alexa Fluor anti-mouse 488. Antibody information is provided in Supplementary file 2.