Involvement of Socs3 protein in the persistent activation of Stat3 in Gprc5a−/− cells. A, Gprc5a+/+ and Gprc5a−/− cells were starved for 48 hours and then their total mRNA was extracted and analyzed for Socs3 mRNA by QPCR (left) and their total protein was analyzed for Socs3 protein by immunoblotting (right). B, cells were treated with 10 µM MG132 for different times and analyzed by immunoblotting. C, left, Gprc5a−/− cells transfected with vector or SOCS3-HA were starved for 48 hours then extracted for protein analysis by immunoblotting. Right, Gprc5a−/− cells transfected with vector or SOCS3-HA were treated with exogenous Lif (1000 units/ml) in K-SFM. After the indicated times, cells were harvested and analyzed for Stat3 activation by immunoblotting. D, left, HEK293T cells transfected with the indicated vectors were cultured for 48 hours and then analyzed by immunoblotting. Middle, HEK293T cells transfected with the indicated vectors for 48 hours were treated with 20 µM cycloheximide for different times and then the cells were analyzed by immunoblotting. Right, Densitometry results for the western blotting bands in the middle panel were plotted.