Sequences required for the interaction of TBLR1 with SMRT. a. TBLR1 constructs were cloned in-frame into pCDNA3 with an N-terminal Flag tag. b. HuSMRT cDNA (1-900) was PCR-amplified and cloned in-frame into PGEX-6p-1. EcoR1 linearized plasmids were purified and used as a template for in vitro translation using 35S methionine. Expression of the GST fusion protein was induced with IPTG and the product adsorbed onto Glutathione sepharose 4B. For the pull-down experiment, 5 ng of 35S labeled and 20 ul of GST agarose were mixed. After incubation the bound material was eluted and electrophoresed through a 10% SDS PAGE gel. 10% of the labeled material was reserved and electrophoresed separately to demonstrate the efficiency of the original labeling.
Zhang et al. BMC Cell Biology 2006 7:31 doi:10.1186/1471-2121-7-31