Figure 3

Western blots of isolated sea urchin vaults. (A) Peptide amino acid sequence of rat MVP (aa 19–35) aligned with Sp MVP (25–41). Anti-peptide antibodies were generated against the rat MVP sequence shown and affinity-purified as previously described [38]. Twelve of the rat MVP amino acids are conserved in the Sp MVP peptide sequence. (B) 150 μg of sea urchin egg extract proteins (lane E) and 10 μg of purified sea urchin vault proteins (lane V) were separated on this Coomassie-blue stained SDS-8% polyacrylamide mini-gel. The arrow shows the position of the 100 kDa MVP in (B, C and D). (C) Alkaline-phosphatase stained western blot showing the migration of the pre-stained protein ladder (lane M: 176.5, 113.7, 80.9, 63.8 (pink), 49.5, and 8.4 kDa polypeptides). Affinity-purified anti-sea urchin MVP antibodies [16] recognize a 100 kDa polypeptide in egg extracts (lane E) and in purified vault preparations (lane V). Asterisks indicate two bands of approximately 80 kDa and 50 kDa that may be breakdown products of the MVP. (D) Affinity-purified anti-peptide antibodies (anti-LDQN, [38]) bind to the 100 kDa MVP polypeptide in purified sea urchin vaults. This peptide antibody appears to bind non-specifically to a large number of polypeptides in the sea urchin egg extracts.