FIGURE 8.
Lack of dimerization does not affect partitioning of MT6-MMP into lipid rafts. A and B, HCT-116 colon cancer cells were stably transfected to express wild type (WT) MT6-MMP (lanes 2 and 2′), or single C532A (lanes 3 and 3′) or triple C530A/C2A/C4A (lanes 4 and 4′) MT6-MMP mutants. Control cells were stably transfected with pcDNA vector without insert (EV) (lanes 1 and 1′). Lysates of stable-pooled populations were resolved by non-reducing (-β-ME) or reducing (+β-ME) 10% SDS-PAGE and examined for expression of MT6-MMP forms by immunoblot analyses. The blots were reprobed with an antibody to the transferrin receptor (TrfR). C, HCT-116 cells stably expressing wild type MT6-MMP (WT) or single C532A or triple C530A/C2A/C4A MT6-MMP mutants were harvested, and cellular proteins were separated into Triton X-100 soluble (S) and Triton X-100-insoluble/β-octylglucoside-soluble (I) fractions. Equal amounts of protein (10 μg/lane) from each fraction were mixed with Laemmli SDS-sample buffer with β-ME and resolved by 7.5% SDS-PAGE followed by immunoblot analysis with mAb MAB1142. The blots were reprobed with an anti-caveolin pAb to detect the ∼22–24-kDa caveolin (Cav). MT6D, MT6-MMP dimeric form; MT6M, MT6-MMP monomeric form.