Fig. 5. (A) Cav-1 and Cav-2 form high molecular weight complexes in Caco-2 cells. Caco-2 cells expressing Cav-1, Cav-2 or both, were lyzed, loaded on top of a 5-40% sucrose gradient and centrifuged for 16 hours at 100,000 g. Top to bottom fractions were TCA precipitated, analyzed by SDS-PAGE and immunoblotted with polyclonal caveolin-1 antibodies (anti-Cav-1) or monoclonal antimyc to detect myc-tagged Cav-2. When expressed alone, Cav-1 was present in high molecular weight complexes while Cav-2 alone was not. Co-expression of Cav-1 along with Cav-2 recruited some Cav-2 and increased the proportion of Cav-1 in high molecular weight complexes. (B) Cav-1 and Cav-2 co-immunoprecipitation in Caco-2 cells. Cav-2 and Cav-1+2 Caco-2 cells were pulse-labeled with ³⁵S methionine for 3 hours and lyzed in 1% Triton X-100. Lysates were immunoprecipitated with polyclonal Cav-1 antibody (1) or mAb65 anti-Cav-2 (2). Immunoprecipitates were analyzed by SDS-PAGE and fluorography. Cav-1 was detected in Cav-2 immunoprecipitates in Caco-2 cells expressing both proteins.