Lipoproteins are necessary and sufficient for secretion of lipoprotein-associated SHH from adrenocortical carcinoma cells in vitro. (A) Secreted SHH from NCI-H295R cells associates with lipoproteins. (B) Western blot of cell lysates and conditioned medium from NCI-H295R cells cultured in serum-free conditions or with 0.625% or 2.5% human serum lipoproteins (HSLpp), probed for SHH. Equal amounts of cell lysate and corresponding conditioned medium were loaded. ACTIN was used as a loading control for the cell lysates. (C) Western blots of density gradient fractions of conditioned medium from NCI-H295R cells cultured with human serum lipoproteins, probed for SHH, APOA1, or APOE. The density of lipoprotein classes is according to Jonas and Phillips (2008). (D) Western blots of immunoprecipitation with antibodies to apolipoproteins APOA1 and APOE from conditioned medium of NCI-H295R cells grown with human serum lipoproteins, probed for the corresponding immunoprecipitated (IP) protein and SHH. Ft, flow-through.