SDS-PAGE analysis of a sample obtained from the column immobilized with the full-length construct

C176 revealed the presence of the 25-kDa band that comigrated with a protein present in the HDL marker (Fig. 1b). In addition, a similar protein band was present in the sample eluted from the column immobilized with C176V, containing the entire noncollagenous V region of Scl1, but not with the truncated construct C176T. This protein-band was absent in control lane (No rScl1). In order to verify that the 25-kDa protein was ApoA I, the same samples were blotted onto a membrane and immunoreacted with specific anti-ApoA I antibodies (Fig. 1c). As expected, the 25-kDa band found in C176 and C176V samples was identified as ApoA I. To confirm the ligand-binding ability of C176 derivatives that were detected using human plasma, we used DAPT in vivo the same affinity chromatography columns with purified HDL. The samples eluted Nutlin 3a from the columns with immobilized rScl1 or PBS were analyzed by 15% SDS-PAGE and Western immunoblotting (Fig. 2). The 25-kDa band of ApoAI contained in HDL was detected in the C176 sample by staining and with the anti-ApoAI antibody, but not in a sample eluted from the control column

without the rScl1 protein. The N-terminal 42-aa-truncated variant of C176 (C176T) was not able to bind to HDL. On the contrary, the recombinant C176V, which contains all 84 amino acids of the V region, but lacks the CL region, could bind HDL, implying that the V region was responsible for the binding. Altogether, our results identified HDL as a new ligand for the Scl1.41

protein. The binding occurs via a noncollagenous domain of Scl1, which is necessary and sufficient for HDL binding. In contrast to P176-LDL binding (Han et al., 2006a), the binding between C176 and HDL could not be detected by traditional ELISA. We hypothesized that the presence of a nonionic detergent, Tween 20, in the wash buffer affected C176-HDL binding. To test this hypothesis, binding experiments using both affinity chromatography and ELISA were performed with or without Tween 20 (Fig. 3). In affinity chromatography analysis, the HDL-binding positive constructs C176 and C176V were immobilized onto duplicate columns enough with Strep-Tactin Sepharose, and purified HDL was passed over the columns. Columns were washed using buffer W with or without 0.05% Tween 20. The eluted samples obtained from affinity chromatography columns treated with Tween 20 did not contain HDL, whereas those without Tween 20 did (Fig. 3a and b). These data were further confirmed by ELISA (Fig. 3c). Microplate wells were immobilized with different concentrations of C176V and incubated with purified HDL. Wells were washed with a buffer containing (TBST) or lacking (TBS) Tween 20 and bound HDL was detected with the anti-ApoAI antibody. The C176V protein was able to bind to HDL in a concentration-dependent manner, indicating that binding was specific, but only when washing was performed with TBS.