Immunoblotting of Irradiated SHH-MB Tumors

SHH-MB tissue was dissected from tumour-bearing mice following 2 Gy irradiation and homogenized through tissue sonication in tissue lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 100 mM NaF, 2 mM Na₃VO₄, 10 mM Na₄P₂O₇) supplemented with protease inhibitor (Sigma, no. P8849). Protein concentration was determined using the Pierce BCA protein assay kit (ThermoFisher), and proteins were separated by NuPAGE Novex 10% Bis-Tris precast gels (ThermoFisher) before electrophoretic transfer to polyvinylidene difluoride membranes. Membranes were incubated first with Odyssey Blocking Buffer (LI-COR Biosciences) and then with primary antibodies for P-selectin, p53 and GAPDH and the appropriate secondary antibodies, and analysed using near-infrared imaging with the LI-COR Odyssey CLx Imaging System. The antibodies used are described in Supplementary Table 2.