CD63 and proNE form high-molecular-weight complexes. (A-F) Cell lysates in RIPA from COS cells transfected with NE, GFP-CD63 (CD63), and NE plus GFP-CD63 as indicated were separated on a Superose 6 column eluted with RIPA. The fractions were analyzed by IP/Western as follows: (A) anti-cproNE/anti-cproNE, (B) anti-GFP/anti-GFP, (C) anti-cproNE/anti-cproNE, (D) anti-GFP/anti-GFP, (E) anti-GFP/anti-cproNE, and (F) anti-cproNE/anti-GFP. The samples were split (empty column) as there were too many for a single gel. (G-I) Cell lysates were examined by IP, SDS-PAGE, and silver staining as indicated. (G) IP with anti-cproNE of cells expressing NE showed stained protein corresponding both to proNE monomer and proNE complex (
). (H) IP with anti-cproNE of cells coexpressing NE and GFP-CD63 also showed stained protein corresponding to proNE monomer and complex (), and copurification of a protein corresponding to GFP-CD63 (→). (I) IP with anti-GFP showed a broad elution profile of a stained protein corresponding to GFP-CD63 (→) as in panel B, but copurification of proNE was barely visible. Arrows show the void volume (V0) and elution volumes for thyroglobulin (669 kDa), ferritin (440 kDa), bovine serum albumin (67 kDa), and trypsin inhibitor (20 kDa).
