Measuring the rate of endocytosis of surface biotinylated FcRn in HepG2 cells.

<p>(A) Cells were surface labelled with sulfo-NHS-S-S-biotin, incubated at 37°C for 0-60min. Surface biotin was stripped from protein at indicated time with a cell impermeable reducing agent. Cells were lysed and incubated with NeutrAvidin beads. The beads were washed and proteins were eluted using DTT and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and immunoblotted for FcRn. The anti-FcRn immunoblot shows the NeutrAvidin bead eluates. To ensure the amounts of biotin were not a limiting factor in surface labelling, sulfo-NHS-S-S-biotin at 0.5mg/ml and 5mg/ml was used (lane 2 and 3). Lane 4 is the strip control (0 min internalisation) to show the efficiency of stripping biotin from surface exposed FcRn. Lanes 5–9 are samples that have been incubated for 5, 10, 20, 30, 60 min respectively and show internal biotinylated FcRn that is resistant to surface stripping. (B) Quantification of biotinylated FcRn that is resistant to surface stripping at indicated time points. The mean ± standard deviation of four independent experiments are shown. No significant difference is seen between5 and 60 min as analysed by linear regression.