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Fig. 2. BBS6 is enriched in centrosomal fractions and its minor soluble form does not associate with CCT or oligomerize. (A) BBS6 is highly enriched in centrosomes purified from NIH 3T3 cells. HeLa cells were either not transfected or transfected with pCMV-Myc-BBS6 or pCMV-BBS6, and total cell lysates were probed by western blot analysis with an affinity-purified polyclonal BBS6 antibody. Wild-type (60 kDa) and myc-tagged BBS6 (62 kDa) can be observed in the transfected cells. (B) Most of BBS6 occurs as a high-MW Triton X-insoluble form distinct from that of cytosolic CCT. Total lysates of IMCD3 cells expressing pCMV-BBS6 were loaded on discontinuous 4-71% sucrose gradient (the top, bottom and sucrose steps in the gradient are indicated). After centrifugation, each fraction was analyzed on a 10% SDS polyacrylamide gel and subjected to western blot analyses with antibodies to BBS6, CCT or -tubulin, as indicated. Most of the BBS6 and -tubulin is found in the 71% and pellet fractions, while the majority of CCT forms a peak at 24% sucrose. Schematics below the panels represent potential positions of monomeric/dimeric or oligomeric chaperonin species. (C) BBS6 does not form homo-oligomers. Extracts of COS-7 cells transiently expressing different constructs (i.e. pEGFP-BBS6 or pCMV-Myc-BBS6, as indicated), or untransfected, were immunoprecitated (IP) with antibodies to myc, GFP or CCT. Lysates (L), immune supernatants (S) or immune pellets (P) were subjected to western blot analyses using antibodies against BBS6, myc, GFP or CCT (UM1 antibody), as indicated. The respective positions of myc-BBS6, GFP-BBS6 and CCT subunits are noted.

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