Figure 5

mbGluc Is also Present as a Free Protein, Independent of Lipid Carriers. (A) Fast protein liquid chromatography (FPLC) of conditioned medium derived from OVCAR5-mbGluc+, OVCAR5-Gluc+ (Gluc that is not fused to transmembrane ___domain and is secreted freely into extracellular space), non-conditioned medium with human HDL spiked-in (HDL-A and HDL-B are replicates of samples with HDL) as well as OVCAR5-mbGluc+ and OVCAR5-Gluc+ media mixed with HDL. Left panel – all fractions, right panel – fractions 1–21 with reduced scale on Y axis. (B) Composition of human HDL fractionated by FPLC, TC – total cholesterol, TG – triglycerides, PC – phosphatydylcholine (data representative of 2 experiments). (C) Western blot analysis of apolipoprotein A-I (ApoA-I) protein (marker of HDL) following fractionation of OVCAR5 conditioned medium. The white bar divides lanes from separate membranes with samples from the same experiment processed according to the same procedure. Full picture of the gel is presented in Fig. S5G. (D) mbGluc bioluminescent activity in structures captured by anti-ApoA-I, anti-Gluc or IgG (unspecific binding) antibody in top fraction of sucrose density-step gradient following fractionation of OVCAR5-mbGluc+ medium. (E) Composition of OVCAR5-mbGluc+ conditioned medium (upper panel), OVCAR5-mbGluc+ conditioned medium with addition of HDL (middle panel) and OVCAR5-mbGluc+ preconditioned medium mixed with HDL in the absence of cells (lower panel) in FPLC fractions. (F) FPLC fractionation of standard proteins to determine the fractionation and distribution of high-molecular weight standards. Zone I corresponds to particles of approximately 25–75 nm in diameter (e.g. VLDL/LDL/small EVs), Zone II corresponds to particles of approximately 7–12 nm in diameter (e.g. HDL) and Zone III corresponds to particles smaller than 7 nm in diameter.