. Doxycycline-inducible shRNAs directed against Hmga1 or GFP were stably expressed in MEFs. Forty eight hours after addition of doxycycline (100 ng/ml), cycloheximide (250 μM) was added and extracts were prepared before and at 2, 4 and 8 hr thereafter. The pre-CHX extracts were immunoblotted for HMGA1 and all samples were immunobloted for Grb14 and rpL26; a representative immunoblot is shown. The relative abundance of Grb14 at 2, 4 and 8 hr, estimated by densitometry, was divided into the preCHX value and the time to a 50% reduction was calculated as 2.47 hr for shRNA-GFP and 21.86 hr for shRNA-Hmga1.