Biochemical study of MEG-01 dense granules. (A) Immunoblotting analysis of fractions obtained from MEG-01 postnuclear supernatants subjected to subcellular fractionation with a 10% to 60% sucrose gradient. Markers for different cell compartments are as follow: MRP4 and LAMP2 for immature and mature DGs; PF-4 for α granules; and Rab7a and Rab32 for cytosol, vesicles, and DGs. (B) ADP (μM, solid lines) and fluorescence intensity of both infrared fluorescent-dextran (A.U., dashed line) and the fluorescent Ca²⁺ indicator Oregon Green BAPTA-1 dextran (A.U., dotted line) in fractions from panel A. The concentration of ADP was determined both in the untreated sucrose gradient fractions (solid circles) and in sucrose gradient fractions enriched in MRP4 structures by immunoprecipitation using an MRP4 antibody (open circles). (C) Sucrose gradient fractions were immunoprecipitated using a Rab38 antibody and the presence of MRP4 in the precipitated structures was determined by immunoblotting, confirming the occurrence of the proteins in the same structures. (D) The coexistence of both LAMP2 and MRP4 in the same organelles was confirmed by coimmnunoprecipitation using a MRP4 antibody (left) or a LAMP2 antibody (right) and immunoblotting analysis using an antibody against the other protein. SGF indicates sucrose gradient fraction; IP, immunoprecipitation; IB, immunoblotting; and IDG, immature DG.