Figure 3.

The A_2AR is a potential substrate of CtsD.A, lysates from HEK-293-FLAG-A_2AR^SNAP cells were incubated in the absence and presence of recombinant mouse CtsD (1.5 μg/ml) for 1 and 2 h. The specific bands were detected by WB using FLAG-tag and β-actin-specific antibodies. Densitometry analysis data are presented as mean ± SD of three independent experiments. Values from ANOVA: F = 3.385 and p = 0.0298. ∗p < 0.05 versus control (in the absence of recombinant mouse CtsD). B, 200 ng of recombinant GST-A_2AR^284–410 and GST proteins were incubated in the absence and presence of recombinant mouse CtsD (10 μg/ml) for 5, 10, and 30 min. The bands of the recombinant proteins were detected in polyacrylamide gel by silver staining. St denotes Precision Plus Protein Dual Color Standards. A_2AR, adenosine A_2A receptor; CtsD, cathepsin D; GST, glutathione-S-transferase; HEK-293, human embryonic kidney 293 cell line; WB, Western blot.