PSMA associates with Lubrol WX-DRMs as a dimeric protein.

<p>(A) LNCaP cells were solubilized with either 1% Lubrol WX or 1% Triton X-100 (w/v) in PBS. Lysates were subjected to centrifugation at 100.000×g for 90 min in order to separate the insoluble (P) DRM-fraction and the soluble (S) material. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and equal amounts of total protein from both fractions were loaded for each lane on SDS gels and blotted. The figure shows that PSMA is completely soluble in Triton X-100 and is partially insoluble in Lubrol WX. To verify these DRMs flotillin-2 and caveolin-1 were used as raft marker proteins and the early endosomal antigen 1 (EEA1) was utilized as a non-raft marker. P pellet, S supernatant (B) After centrifugation the insoluble fraction (P) was solubilized with 1% (w/v) Triton X-100 and 1 ml of both insoluble (P) and soluble (S) fraction was loaded on top of a 5–25% continuous sucrose gradient. After 18 h of centrifugation at 100.000×g, 24 fractions of 500 µl each were collected from bottom to top. Half of each fraction was precipitated using ethanol and PSMA was detected by western blot analysis. The figure shows monomeric PSMA peaking in fractions 16–19 where the mannose-rich glycosylated form (PSMA_M) can be distinguished from its complex glycosylated counterpart (PSMA_C). Dimers of PSMA are located in fractions 13–15 that exclusively contain complex glycosylated PSMA (PSMA_C). PSMA in Lubrol WX-DRMs migrates like the dimeric form of PSMA whereas detergent-soluble PSMA is distributed in later fractions (16 and 17) that contain monomeric PSMA.