Figure 3 : A. Expression of GR WT, CREB, CREB-serine133, and PPP2R3C in bacteria. The factors were cloned into pRSET vector and fusion proteins were purified for in vitro binding and reconstitution assays. Fusion proteins CREB (lane 1), CREB-133 (a mutant variant of the human CREB protein that contains a serine to alanine mutation corresponding to amino acid 133, lane 2), GRwt (lane 3) and PPP2R3C (lane 4) respectively were induced with IPTG and purified as described Experimental Procedures. The CREB was phosphorylated as described and used for in vitro de-phosphorylation assays. B. De-phosphorylation of CREB-P by GST-GRDEX bound PPP2R3C reconstituted PP2A complexes from T47D extract: CREB-P de-phosphorylation was performed in the absence of 100 nM DEX (Lanes 1-7) and in the presence of DEX (Lanes 8-14). The quantity of phosphorylated CREB was determined by immunoprecipitation using polyclonal anti-CREB-P antibodies and counting the 32P-radioactivity in immunoprecipitates. Experiments were repeated a minimum of three times before plotting the results. Controls without ligand and with ligand treatments are shown for reference. Extracts prepared from T47D cells in the absence of ligand served as control.

Govindan et al.Molecular Biology and Genetic Engineering  2013 1:2DOI : 10.7243/2053-5767-1-2