Goat anti rabbit hrp

Cells were lysed in ice cold RIPA buffer with protease inhibitor for 35 min, and centrifuged at 4 °C, 11,000 g for 15 min; 10 µg protein was subjected to SDS-PAGE and subsequently transferred to a polyvinyl difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were incubated with the primary antibody at 37 °C, for 1 h followed by incubation with the secondary antibody at 37 °C for 1 h. The primary antibodies were N-cadherin (Cat. no 22018-1-AP, Proteintech, USA), E-cadherin (Cat. no 60335–1-lg, Proteintech, USA), Vimentin (Cat. no 10366-1-lg, Proteintech, USA) Snail (Cat. no 13099-1-AP, Proteintech, USA), Slug (Cat. no 12129-1-AP, Proteintech, USA). Secondary antibodies were goat anti-mouse HRP (Cat. no SA00001-1, Proteintech, USA) or goat anti-rabbit HRP (Cat. no SA00001-2, Proteintech, USA). β-actin (Cat.no TA-09; Zsbio, China) was used as a housekeeping gene control.

Western blotting analysis was conducted following the procedure as previously described.³⁹ Briefly, foetal liver samples at indicated developmental stages were homogenized in RIPA buffer (Sigma‐Aldrich) supplemented with Protease/Phosphatase inhibitors (APExBIO). Protein samples (40‐80ug) were separated with 10%‐15% of SDS‐PAGE gel. The following primary antibodies are used in this study: rabbit anti‐Nbs1 (1:1000, Cell Signaling; or a homemade Nbs1 antibody); rabbit anti‐p53 (Ser18) (1:1000, Cell Signaling); mouse anti‐γ‐H2AX (1:5000, Merck Millipore); mouse anti‐Chk2 (1:1500, Merck Millipore); mouse anti‐β‐Actin (1:5000, Proteintech). The secondary antibodies used are goat anti‐rabbit HRP and goat anti‐mouse HRP (1:2500, Proteintech).
For immunofluorescence analysis on DNA double‐strand breaks (DSBs) in HSCs and haematopoietic progenitors (LK progenitors), cells were FACS sorted onto gelatin‐coated coverslips. The subsequent fixation and antibody staining procedure followed the published protocol.⁴³ The mouse anti‐γ‐H2AX antibody (1:800, Merck Millipore) was used to investigate DSBs in each cell population.