Cy5 fluorescent dye

The lectins were labeled with Cy5 fluorescent dye (GE Healthcare, U.S.A) and purified using Sephadex G-25 columns (GE Healthcare, U.S.A). Dewaxed and rehydrated tissue sections were microwaved in 10 mM citrate buffer (pH 6.0) at 100 °C for 10 min and cooled at room temperature to eliminate endogenous peroxidase activity, and 5% bovine serum albumin and 0.08% Triton X-100 was used to block nonspecific staining at 25 °C for 1 h. The tissue sections were incubated with Cy5-labelled lectin at 4 °C overnight. Finally, sections were stained with 1 μg/ml of DAPI (Roche, Switzerland) for 10 min. Cells were pre-inoculated into the dish, and 4% paraformaldehyde was used to immobilize the cells for 10 min. Then 5% bovine serum albumin and 0.01% Triton X-100 were used to block nonspecific staining at 25 °C for 1 h. Cells were incubated with Cy5-labelled lectin at 4 °C overnight. Finally, sections were stained with 1 μg/ml of DAPI for 10 min. A laser scanning confocal microscope FV 1000 (Olympus, Japan) was used to obtain the images. The images were acquired using the same condition and shown on the same scale in the Cy5- and DAPI-merge channel. The average fluorescence intensities of the images were obtained by total fluorescence intensities of lectins binding with cells divided by the total area of cells.

Tapasin and TAPBPR immunoprecipitates were pre-labelled with Cy5 fluorescent dye (GE Healthcare) and subjected to electrophoresis using the Amersham WB system. Cy5 total protein images from four separate electrophoresis experiments were analysed by ImageJ. Densitometry graphs from relevant tracks were subsequently analysed in MATLAB. For the tapasin immunoprecipitate, a MATLAB script was developed (see Source code 1) and applied in order to calculate the densities of the A68 and B15 bands.

Plasma samples from HF and HDF patients (15 μg of protein) were separated and analysed by denaturating sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) of 12% acrylamide (3% cross-linking). Low molecular weight calibration kit (GE Healthcare) for SDS-PAGE was used as molecular mass standards.
In DIGE experiments, plasma samples from HF and HDF patients (5 μg of protein) were labelled with Cy3 or Cy5 fluorescent dyes (GE Healthcare) according to the standard protocol for DIGE assay, as previously described^{21 (link)}. Labelling was performed in samples from HF and HDF patients (n = 9 per group) in a paired combination. A sample of HF (patient 1) was randomly discarded and the labelling was alternated between Cy3 and Cy5 in each group. After fluorescence labelling, HF and HDF samples were combined and analysed by SDS-PAGE as described above. In the Cy-labelled experiments for subsequent MALDI-TOF MS identification pools (n = 5) of both HF and HDF patients’ plasma samples were combined. A detailed protocol is included in the Supplementary Material.
Coomassie brilliant blue (R-250, BioRad) was used to stain the proteins in gels, that after were washed-out with 20% ethanol/7% acetic acid (vol/vol). Finally, gels were preserved in 10% ethanol until scanning and protein quantification, or extraction for subsequent identification by MS.