FIGURE 1.

HO-1 protein-protein interactions revealed by chemical cross-linking and co-immunoprecipitation. A, HEK293 cells were transfected with the indicated amounts of plasmids coding for HO-1-CΔ42 or HO-1-FL for 24 h, followed by cross-linking with DSP, then cell lysates were subjected to SDS-PAGE in the presence or absence of 10 mm dithiothreitol and HO-1 protein examined by Western blot analysis. B, HEK293 cells were transfected with plasmids carrying Flag-HO-1 or/and YFP-HO-1 cDNA for 24 h, then were solubilized with lysis buffer as described under “Experimental Procedures.” Equal amounts of cell lysates were then subjected to immunoprecipitation using an anti-Flag affinity gel. Both crude lysates and immunoprecipitates from cells transfected with YFP-HO-1 cDNA (lanes 1 and 4), Flag-HO-1 cDNA (lanes 2 and 5), or YFP-HO-1 and Flag-HO-1 cDNAs together (lanes 3 and 7) were subjected to Western blotting using anti-HO-1 antibodies to detect the presence of YFP-HO-1 and Flag-HO-1. Lane 6 represents the immunoprecipitate from a mixture of lysates from cells transfected with Flag-HO-1-cDNA or YFP-HO-1-cDNA alone (mixed in a 1:1 ratio).