Pulse‐chase experiments

Newly synthesized proteins were labeled using L‐azidohomoalanine (AHA), which is an analogue of methionine that contains an azide moiety. After pre‐incubation in methionine‐free medium for 30 min, MEFs were incubated for 180 min in methionine‐free medium supplemented with AHA. Free AHA was removed by washing with PBS, and complete medium was added to the MEF cultures. After incubation for 0, 24, 48, 72, and 96 h, MEFs were harvested and lysed with 1% SDS in 50 mm Tris/HCl (pH8.0). AHA incorporated into nascent proteins was labeled with biotin–alkyne by a ‘click’ reaction. Biotinylated lysates (100 μg) and streptavidin beads (JSR, Tokyo, Japan) were incubated at 4 °C for 1 h in a solution containing 0.5% NP‐40, 20 mm Tris/HCl (pH 7.4), 150 mm NaCl, and 0.5 mm DTT. Beads were washed with the same buffer three times, and pull‐down products were eluted by heating with SDS/PAGE sample buffer to perform western blotting.