Protein extraction and western blotting

Cells were initially washed twice with cold PBS and lysed in lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, EDTA 5 mM, NP40 1%, protease and phosphatase inhibitors), followed by centrifugation at 13,000 g for 10 min at 4°C. Protein extracts from mouse tissues were isolated as previously described (Boutant et al., 2014). Cleared protein lysates were quantified using BCA assay (Pierce). For western blotting, proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA prepared in TBST and incubated overnight with antibodies against target proteins (refer to Key Resources Table for information on antibodies and dilutions). Membranes were then developed by enhanced chemiluminescence (Amersham). For quantification, the intensity of each band was determined by densitometry using ImageJ software.