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Fig. 5. Appendage selectivity of the mosquito ARH-like proteins. (A) GST ( $~$ 400 µg, lanes a,b,e,f) or GST-AgARHC1 (residues 196-300; lanes c,d,g,h) immobilized on glutathione-Sepharose was incubated with 100 µg/ml of either thrombin-cleaved ${alpha}$ _C appendage (open arrowheads) or β2 appendage (arrowheads) in the presence of 25 µM PPACK and 100 µg/ml BSA. After centrifugation, 2.5% of each supernatant (S) and 12.5% of each washed pellet (P) were resolved by SDS-PAGE for staining or fivefold less of each supernatant and pellet were transferred onto nitrocellulose. Blots were probed with either anti-AP-2 ${alpha}$ subunit mAb 100/2 or affinity-purified rabbit anti-AP-2 β2 subunit antibodies. (B) GST ( $~$ 250 µg, lanes a,b,e,f) or GST-AaARH (residues 20-292) on glutathione-Sepharose was incubated with either 50 µg/ml thrombin-cleaved ${alpha}$ _C wild type (lanes a-d) or ${alpha}$ _C (Q782A) mutant (lanes e-h) in the presence of 100 µg/ml BSA. After centrifugation, 2.5% of each supernatant (S) and 12.5% of washed pellets (P) were resolved on SDS-PAGE for staining or fivefold less of each supernatant and pellet transferred onto nitrocellulose. The blot was probed with the anti-AP-2 ${alpha}$ subunit mAb 100/2. Stained ${alpha}$ appendage in supernatant (open arrowheads) and pellet (arrowhead) fractions is indicated.

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