Extraction and Analysis of Proteins from Bone Marrow

Proteins from bone marrow samples were extracted as previously described (31 (link)). Tissue was lysed in 500 μl of ice‐cold lysis buffer (40 mM Tris–HCl pH 7.4, 1% Triton X‐100, 40 mM Beta‐glycero phosphate, 5% Glycerol, 100 mM NaCl, 1 mM EDTA, 50 mM NaF and protease and phosphatase inhibitors (PMSF (1 mM), aprotinin (0.019 TIU/ml), leupeptin (1 μg/ml), NaF (5 mM) and Na₃VO₄ (1 mM)) per 100 mg and then crushed using a sterile pestle (Axygen). Lysates were incubated for 30 min on ice and sonicated 30 s ON\OFF for 10 cycles with a Bioruptor (Diagenode). Insoluble material was removed by high‐speed centrifugation at 4°C. Protein extracts were subjected to western blot analysis using anti-FANCI (Bethyl, A301-254A) or anti-FANCD2 (Abcam, ab108928).