Fig. 5. NPP2 is processed by furin-like endoproteinases. (A) The N-terminal sequence of NPP2 with the predicted signal peptide cleavage site (white arrow), the consensus site for recognition by furin (underlined) and the predicted furin cleavage site (grey arrow). (B) HA-NPP2-Myc with the Flag-tag inserted after Arg35 was expressed in HEK293 cells. After 24 hours and 72 hours the cell lysates and culture medium were processed for immunoblotting with the anti-Flag M1 and M2 monoclonal antibodies. (C) The same NPP2 construct was also expressed for 72 hours with or without the furin-inhibitor ₁-PDX, and the corresponding lysates and media were immunoblotted with the M1 and M2 antibodies. (D) HA-NPP2-Myc with a Flag-tag after Arg35 was expressed in ß-TC3 cells before or after the RNAi-mediated knockdown of furin or PACE4, as indicated. Aliquots of the culture medium were processed for immunoblotting with the M1 and M2 antibodies. The right panels show the effect of the furin or PACE4 shRNAs on the level of overexpressed furin or PACE4, respectively, as detected by immunoblotting. The data are representative for at least three independent experiments.