Repeated surface biotinylation.

<p>(A) HepG2 cells were surface labelled with sulfo-NHS-S-S-biotin, followed by incubation at 37°C for 10 min (lane 1–4). Cells (lane 2–4) were re-biotinylated and incubated at 37°C for a further 10 min. Cells (lanes 3–4) were re-biotinylated and incubated for a further 10 min at 37°C once and twice respectively. After incubation at 37°C cells were lysed and incubated with NeutrAvidin beads. The beads were washed, proteins were eluted using DTT and then separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and immunoblotted for FcRn. The anti-FcRn immunoblots shows the whole cell lysates (top panel) and corresponding NeutrAvidin bead eluates (bottom panel). (B) Cells were treated as above except rather than cells being lysed immediately following 37°C incubation they were placed at 4°C and treated with cell impermeable reducing reagent. (C) Quantification of biotinylated FcRn following different treatments. The mean ± standard deviation of three independent experiments are shown. Linear regression analysis reveals significant increase in total biotinylated (p = 0.003) and internalised biotinylated (p = 0.0283) FcRn upon increasing rounds of biotinylation.