Fig. 4. Coat-binding properties of mosquito ARH-like proteins. (A) GST (100 µg, lanes a and b) or GST-AaARH (residues 21-292; lanes c and d) or human GST-ARH (residues 1-308; lanes e and f) on glutathione-Sepharose was incubated with rat brain cytosol. After centrifugation, aliquots of 2% of each supernatant (S) and 12.5% of each washed pellet (P) were resolved by SDS-PAGE and stained or transferred to nitrocellulose. Portions of the blots were probed with the anti-clathrin heavy chain (HC) mAb TD.1 and AP-1/2 β subunit antibody GD/1, or anti-µ2 subunit serum, or anti- subunit mAb clone 8, or anti-clathrin light chain (LC) mAb Cl57.3. Only the relevant portion of each blot is shown. (B) GST (250 µg, lanes a and b), GST-ARHC1 (lanes c and d), GST-AgARHC1 (residues 196-300; lanes e and f), GST-AgARHC1 (LIDAAA) (lanes g and h), or AgARHC1 (W268A) (lanes i and j) on glutathione-Sepharose was incubated with rat brain cytosol. After centrifugation, aliquots of 2.5% of each supernatant (S) and 12.5% of washed pellets (P) were resolved by SDS-PAGE and stained or transferred to nitrocellulose. Portions of the blots were probed with the anti-clathrin HC mAb TD.1 and affinity purified anti-AP-1/2 β subunit antibody GD/1, or anti-µ2 subunit serum or affinity purified anti-µ1 antibody RY/1. Note that only the human ARHC1-fusion protein binds to the µ1 subunit (AP-1) as a consequence of the selectivity for the β-subunit appendage. The mammalian AP-1 β1 and AP-2 β2 subunits are 85% identical and the appendages share binding partners.