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. 2010 Jul 2;285(35):27130–27143. doi: 10.1074/jbc.M110.153213

FIGURE 6.

FIGURE 6.

Matriptase cleaves ASIC1-GFP and GFP-ASIC1, but not ASIC2-GFP, in transfected CHO cells. A, dose response of increasing amounts of matriptase DNA co-transfected in CHO cells with 2 μg of ASIC1-GFP DNA. Transfected CHO cells were lysed 2 days post-transfection, and whole-cell lysates were subjected to Western blot with a GFP antibody. The densitometry of the full-length ASIC1-GFP band shows a linear relationship between increasing amounts of matriptase DNA and decreasing amounts of full-length ASIC1-GFP and no effect of the catalytically inactive matriptase S805A. Data in the densitometry graph are means ± S.E. B, CHO cells transfected with 2 μg of ASIC1-GFP alone (control lanes) or plus 2 μg of matriptase or S805A were lysed after being left untreated or after treatment with the proteasome inhibitor MG132 (5 μg/ml, for 5 h or overnight). The whole-cell lysates were subjected to Western blot with GFP antibody. The amount of full-length ASIC1-GFP decreases when it is co-transfected with matriptase but not with the catalytically inactive matriptase S805A. Two small fragments are barely detected in the untreated samples of the matriptase lane. In the samples treated with MG132, the signal of the fragments in the matriptase lane (F1 and F2) is increased. There are no ASIC1-GFP fragments in ASIC1-GFP or ASIC1-GFP plus matriptase S805A in either the untreated or the MG132-treated samples. Data in the densitometry graph are means ± S.E. C, the same conditions apply as in B, with the exception that GFP-ASIC1 (ASIC1 with an N-terminal GFP tag) was used instead. One fragment of GFP-ASIC1 is detected only in the matriptase lanes, and its intensity increases with MG132 treatment. The densitometry data are means ± S.E. D, the same conditions apply as in B and C, with the exception that ASIC2-GFP was used instead and MG132 treatment was overnight. Even though matriptase decreases the full-length ASIC2-GFP, no ASIC2-GFP fragments appear in the matriptase lanes of untreated or MG-132-treated samples, suggesting that matriptase does not cleave ASIC2-GFP. The densitometry data are means ± S.E. of three separate experiments.