Collection of protein from conditioned medium, preparation of total cell lysates, and western blot analysis

P2 NSCs from each pup were seeded at a density of 100,000 cells ml^–1 in T-25 flasks (Thermo) and maintained under proliferation conditions for 5 days. Neurospheres suspended in conditioned medium were harvested in a 15 ml conical tube and centrifuged. Cell pellets were lysed in RIPA buffer (Sigma) supplemented with Pierce phosphatase inhibitor mini tablets (Pierce) and cOmplete protease inhibitor cocktail (Roche) on ice for 20 min and sonicated for 20 s. Lysates were centrifuged at 13,000 × g for 5 min at 4 °C. Supernatant was collected in screw capped 2 ml tubes, snap frozen in liquid nitrogen, and stored at –80 °C. Conditioned medium was further centrifuged and filtered (0.22 µm pores), snap frozen in liquid nitrogen, and stored at –80 °C. The concentration of protein in the conditioned medium was measured using the Bio-Rad Protein assay (Bio-Rad). For each sample, 150 µg of protein was loaded onto a 15% or 4–20% gradient SDS polyacrylamide gel after boiling in Laemmli buffer (Sigma). Protein bands were transferred to a nitrocellulose membrane, which was then blocked in 2% bovine serum albumin (BSA) in filtered 1 x TBS buffer (36 mM Tris Base, 50 mM NaCl and 0.5% Tween 20). The membrane was then incubated with rabbit polyclonal anti-Igfbp2 (Millipore, 1:1000), goat polyclonal anti-Igfbp2 (R & D systems, 1:500), rabbit monoclonal anti-γ-H2ax (Abcam, 1:1000), goat polyclonal anti-Gapdh (ThermoFisher, 1:1000), Rad51 (mouse, 1:1000, Abcam), Nestin (chicken, 1:750, Novus Biologicals), or mouse monoclonal anti-Flag (Sigma, 1:500) in 0.5% BSA in filtered 1 x TBS, overnight at 4 °C. The membrane was then washed and incubated in 0.5% BSA in filtered 1 x TBS containing IRDye 680 donkey-anti-rabbit, IRDye 800 donkey-anti-goat antibody (1:10,000, LI-COR Biosciences), Alexa Fluor 488-Conjugated Donkey anti-chicken (Jackson ImmunoResearch, 1:10,000), or HRP-Conjugated Donkey anti-mouse (Jackson ImmunoResearch, 1:10,000) for 2 h at room temperature. The membrane was then imaged using the Odyssey or Odyssey M imaging system (LI-CORE Biosciences). Densitometry was carried out using the Image Studio Lite software and Empiria Studio (LI-CORE Biosciences).