Carboxylink coupling gel

Preparation of cholesterol beads was based on a previous report45 (link)46 (link) and the manufacturer’s instructions. CarboxyLink coupling gel (Thermo Scientific) was washed in 30% 1,4-dioxane (Fisher) and then in 100% dioxane. Cholesteryl hemisuccinate (Sigma, C6512) was dissolved in 100% dioxane, and immobilized on a pre-washed CarboxyLink coupling gel in the presence of N,N’-dicyclohexylcarbodiimide (DCC; Thermo Scientific, 20320) at room temperature for 4 hours. The cholesterol-beads were then washed twice each in 100% dioxane, 30% dioxane, and then stored in PBS. At the same time, pre-washed CarboxyLink coupling gel was processed without Cholesteryl hemisuccinate to serve as a control for non-specific binding. Cholesterol-beads or unconjugated control beads were mixed with cell lysates containing membrane fractions isolated from stable Sf9 cells (described above) in the presence or absence of 1 mM cholesterol, and incubated for 1 h at room temperature. The reaction mix was then centrifuged and washed 5 times with 50 mM Tris, and eluted with 2X Laemmli Sample Buffer (BioRad) at room temperature for 30 min. Eluates were analyzed by Western blotting.

To generate the N protein aptamer, an N protein affinity column was prepared using CarboxyLink^TM Coupling gel (Thermo Fisher Scientific Inc, USA) in accordance with the product manual. In brief, a 2 mL portion of coupling gel was introduced into the column, washed with 10 mL of 0.1 M MES buffer (pH 4.7) containing 0.9% NaCl. Then, 1 mL of N protein solution (1 mg dissolved in 1 mL of 0.1 MES buffer) and 1-Ethyl-3-[3-dimethylami-nopropyl]-carbodiimide hydrochloride solution (EDC, 1 mg dissolved in 0.5 mL of 0.1 MES buffer) were added. The reaction was allowed to proceed overnight at 4 °C. After passing the reaction mixture through the column, any unbound N protein was removed by washing with 20 mL of 1 M NaCl solution. Finally, the N protein affinity column was stored in 20% ethanol until it was ready for use.

Photofrin (100 μg) was mixed and incubated with 2.5 μl EDC (1-ethyl-3-[3-dimethylamino-propyl] carbodiimide hydrochloride; Thermo Scientific) 120 mg/ml in 0.1 M MES buffer, pH 4.7, 0.45 mg NHS (N-hydroxy-succinimide; Thermo Scientific) and 10 μl CarboxyLink^TM coupling gel (Thermo Scientific) at room temperature for 3 h. The beads were then washed six times with PBS and used as Photofrin-coupled beads. Photofrin-coupled or control beads were incubated with 1 mg A431 cell lysate at 4 °C for 3 h, supernatants were collected, and the beads were washed three times with 1 M NaCl and three times with PBS. The washed beads were divided into two portions. The first was subjected to SDS-PAGE. The second was subjected to reduction/alkylation by DTT/IAA and eluted by 6 M guanidine hydrochloride (GndCl; Sigma-Aldrich). The eluted proteins were diluted 20-fold with 25 mM ammonium bicarbonate to a final concentration of 0.2 M GndCl, and then digested overnight with trypsin (enzyme: substrate = 1:50). The resulting tryptic peptides were desalted with C18 Z tips (Millipore) and subjected to dimethyl labeling for quantitative analysis (see below).