GST pull-down and immunoprecipitation assays.
CHAPS-solubilized retinal extracts, expression, and purification of GST-fused constructs were prepared as previously described [65]. GST pull-down assays were carried out with 0.5–2.2 μM of GST-fused proteins [65]. Co-precipitates were resolved on SDS-PAGE and analyzed by Western blot with antibodies described in the Results section. Unfolded and partially denatured Cox11 were generated by incubating recombinant and native Cox11 overnight with 5.6 and 7 M guanidine hydrochloride and urea, respectively. The Cox11 conformers were then diluted ~20-fold in CHAPS-binding buffer containing GST-LD. Immunoprecipitation assays with 5 μg of antibody and Western blots (~ 200–400 ng/ml of antibody) were performed exactly as described previously [13].