EcR-GR cells were supplemented with 1 μM sodium selenite and treated with EtOH as a vehicle control, 10 μM ponasterone A, 10 nM dexamethasone, or a combination of both for 24 hr. SelP was partially purified from the culture medium of these cells using Ni-NTA agarose. Culture medium was mixed with the Ni-NTA agarose and the mixture was incubated on a nutating mixer at 4°C overnight. The Ni-NTA beads, along with any bound proteins, were collected by centrifugation, washed twice with 500 μl cold PBS, and then mixed with loading buffer and separated by NuPAGE 4–12% Bis-Tris gels. Proteins were transferred to a polyvinyl difluoride membrane. Membranes were blocked with 5% nonfat dry milk in TBS-T and then probed for SelP (antibody specific for SelP was a gift from Drs. Kris Hill & Raymond Burk, Vanderbilt University). A peroxidase conjugated secondary antibody was used to detect chemiluminescence indicative of protein expression.
