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. 2018 May 30;8:183. doi: 10.3389/fcimb.2018.00183

Figure 6.

Figure 6

PI15 supports initial CPAF activation. (A) Effect of PI15 on CPAF protease activity was studied in vitro. 60 μg of total cell extract (CE) from HeLa cells were incubated with 1 μM recombinant CPAF zymogen lacking the signal peptide (CPAFz−SP) for 30 min at room temperature in absence or presence of increasing concentrations of recombinant PI15 (rPI15). CPAF protease activity was measured by detecting the CPAF substrates Cytokeratin-8 (CK8) and RFX5 by immunoblotting. SMN and actin were tested as controls as they are not affected by CPAF protease activity and therefore served as loading controls. (B) Unrelated protein. (B) Both CK8 and RFX-5 protein bands were quantified from the above blot. Quantification results from 3 independent experiments were plotted as a bar graph. ** ≤ 0.005 (C) PI15 supports the initiation of CPAF activation. Recombinant CPAF zymogen carrying the signal peptide sequences (CPAFz+SP) was incubated with different concentrations of recombinant PI15 (rPI15) for 30 min at room temperature. CPAF and PI15 were detected by immunoblotting.