Figure 3. W287G and Y71G show increased resistance to tryptic cleavage.

CHO cells were transfected with wild-type, W287G or Y71G ASIC1a. Following biotinylation of surface proteins, cells were treated with 0, 1, or 10 µg/ml trypsin for 20 mins. Surface proteins were isolated and blotted for ASIC1a. Note that wild-type ASIC1a at the surface was cleaved by trypsin in a dosage-dependent manner. Also note that the amount of uncleaved mutants was comparable to that of uncleaved WT, although the mutants only had a small fraction of cleaved population (see the bottom image with level enhanced). The decrease in loading in trypsin treated lanes was likely due to the loss of some cells during the treatment. Blots shown are representative from three separate experiments.