Fig. 7
From: A human endothelial cell-based recycling assay for screening of FcRn targeted molecules
HERA screening of HSA variants. Recycling of WT HSA at a pH 7.4 and b pH 6.0 after 4 h incubation with titrated HSA concentrations (100–3200 nM). c An illustration of the crystal structure of HSA with the amino acid residues targeted by mutagenesis (K500A and K573P) highlighted in red spheres. The figure was designed using the PyMOL software with the crystallographic data of HSA70. Histograms showing relative recycling of d WT HSA and K500A, e HSA and MSA, f WT HSA by HMEC1-FcRn cells transfected with a control or hFcRn-specific siRNAs. Relative recycling of WT HSA in the absence or presence of either g ADM31 (binds the HSA binding site on hFcRn), h DVN24 (binds the IgG binding site on hFcRn) or i ADM12 (does not bind the ligand binding sites). MicroScale Thermophoresis measurements where j WT HSA-EGFP and k HSA-K573P-EGFP at a constant concentration (100 nM) were added to titrated amounts of hFcRn at pH 5.5 and 7.4. Binding data were derived from the specific change in thermophoretic mobility and the ratio of normalized time-averaged (1 s) fluorescence intensities at defined time points of the MicroScale Thermophoresis traces (−1 and 4 s). The data from three independent experiments with three replicates are shown where error bars show ± s.d. l Analytical hFcRn affinity chromatography of WT and K573P. The elution profiles are shown as relative fluorescence intensity and as a function of a pH gradient. m Uptake, n recycling and o residual amounts when equal amounts of WT and K573P were added at pH 7.4. The amounts of HSA variants were quantified using ELISA. The obtained data are shown as mean ± s.d. of two independent experiments performed in triplicates
