CD14 endocytosis and shedding

A biotinylation-based assay was used to assess CD14 endocytosis essentially as described earlier [22]. Briefly, Raw264 cells (1.5 × 10⁶ per 3.5-cm dish) were grown for 24 h, the culture medium was exchanged for a fresh one, and after 2 h the cells were washed and overlaid with ice-cold PBS containing 1 mM MgCl₂ and 0.1 mM CaCl₂, pH 8.2 (PBS+) supplemented with 0.5 mg/ml EZLink Sulfo-NHS-SS-Biotin (30 min on ice; Thermo Fisher Scientific). Subsequently, the excess of biotin was removed by washing the cells with PBS + containing 5% FBS and 25 mM lysine, and twice with ice-cold PBS+. At this stage of the procedure one of the samples of control and flotillin-depleted cells was lysed (30 min on ice) in 300 μl of a lysis buffer (25 mM HEPES, 0.5% SDS, 0.5% NP-40, 10% glycerol; pH 8.2) containing inhibitors (1 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mM Na₃VO₄, 50 μM PAO, 10 mM p-nitrophenyl phosphate) and 250 units of Benzonase (Merck). An aliquot of 20 μl was withdrawn from each lysate for 10% SDS-PAGE and immunoblotting analysis of the input CD14 (“input 0 min”), while the rest served to determine the level of surface-bound CD14, as described below. The other samples were incubated in a complete culture medium for 30, 60, or 90 min at 37 °C to induce endocytosis. To remove the biotin tag remaining on the cell surface, the cells were washed with PBS + and incubated twice (15 min each) with 50 mM MESNA (Merck) in 100 mM Tris, 100 mM NaCl, pH 8.6, followed by a wash with 5 mg/ml iodoacetamide in PBS+. Finally, the cells were washed with PBS + and lysed (30 min on ice) in 300 μl of the lysis buffer. An aliquot of 20 μl was withdrawn from each lysate to analyze the input CD14 as above (“input 30–90 min”). The remaining portion of these lysates, as well as of the lysates obtained after cell surface biotinylation, was diluted 5 times in the lysis buffer without detergents and incubated with streptavidin beads allowing isolation of internalized and cell surface proteins, respectively. For the incubation (overnight at 4 °C with agitation) 30 μl of the beads per sample was used (High Capacity Streptavidin Resin, Thermo Fisher Scientific). Next, the beads were washed three times with the lysis buffer containing 0.1% SDS and 0.1% NP-40, and bound proteins were eluted by incubation for 10 min at 95 °C in 70 μl 2 × SDS-sample buffer containing 4% β-mercaptoethanol, and the released proteins were subjected to 10% SDS-PAGE and immunoblotting. At 30–90 min of endocytosis, culture supernatants were collected, soluble biotinylated proteins were adsorbed on streptavidin beads and processed as above for immunoblotting detection of sCD14 shed from the cell surface.