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Fig. 3. The cytosolic domain of CD317 binds the AP1 and AP2 adaptor-complex subunits µ1 and µ2. (A) Immunoblots, probed with an anti-His tag antibody, of pull-down assays between a biotinylated N-terminal peptide of CD317 and His-tagged thioredoxin (His-TRX)-fusion proteins of the µ subunits of the AP1 and AP2 adaptor complexes or LDH. Lanes are labelled according to the loaded lysate (10% of input for pull-down); biotin-labelled lanes represent material isolated using biotin-coated beads and CD317-labelled lanes material isolated using beads coated with biotinylated CD317 peptide, pep1 and pep2 refer to specific and non-specific competing peptides. Markers indicate molecular mass in kDa. (B) Immunoblots of lysates from HeLa cells that had been transfected with siRNA targeting µ2 (µ2 siRNA) or siRNA targeting lamin A/C (lamin A/C siRNA) and probed with anti-µ2 or anti-lamin antibodies as indicated. Blots were then stripped and reprobed with an anti-tubulin antibody as a loading control. (C) HeLa cells that had been transfected with siRNA targeting µ2 or siRNA targeting lamin A/C (as indicated) were used for uptake of transferrin, EGF or CD317 antibody for 15 minutes and then acid-washed to remove any non-internalised material. Bars, 10 µm. (D) Quantification of the data presented in C. PI/cell, pixel intensity per cell. Transferrin uptake in µ2-knockdown cells is 8% of control (n=106 and 168), CD317 uptake in µ2-knockdown cells is 15.2% of control (n=20 and 21), EGF uptake is not affected (n=118 and 92).

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