Figure 4

RXR, RAR and E2F-1 proteins form complexes with hNPDC-1 In Vivo PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Subsequently clarified cell lysates were prepared, and incubated for with a 50% slurry of Trx-S-hNPDC beads (bacterially expressed hNPDC fused at its carboxyl terminus with S-peptide and thioredoxin tags and affinity purified by binding to S-protein agarose beads as described in the Methods section). Ligands, atRA and 9cRA were diluted to a concentration of 1 μM into clarified lysate before the addition hNPDC. Beads were collected, washed, bound protein was eluted from beads with 2X SDS electrophoresis sample buffer and proteins were separated on 12.5% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and probed for the presence of HA-tagged E2F-1, RARβ, or RXRα••••••••••••••