Figure 6. Co-immunoprecipitation of Hsp60 and N-SMase2 in N-SMase2–transfected HEK293 cells.

The cells were seeded into six-well dishes, and 24 h later, they were transfected with N-SMase2 plasmids (2 µg/well). After 48 h, harvested cells were resuspended in PBS with or without DSP. Hsp60 and N-SMase2 were immunoprecipitated from cell lysates, as described under “Experimental Procedures.” Aliquots of the immunoprecipitates were analyzed for Hsp60 and N-SMase2 content by SDS-PAGE and immunoblotting (A). Also N-SMase2-transfected HEK cells were harvested and treated with DSP. N-SMase, Hsp10, Hsp60 and Hsp70 were immunoprecipitated from the cell lysates as described above and the immunoprecipitates were analyzed for N-SMase2 and Hsp60 by immunoblotting (B).