Fig. 3: PRTN3 binds STAT3’s N-terminal domain to identify cleavage sites.

a The schematic diagram of human STAT3 and its truncated mutants. b The schematic diagram of human PRTN3 and its truncated mutants. c Flag-tagged STAT3 or its mutants and HA-tagged PRTN3 were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies (n = 3). d HA-tagged PRTN3 or its mutants and Flag-tagged STAT3 were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with an anti-HA antibody and then immunoblotted with the indicated antibodies (n = 3). e measurements of the binding affinity between the different domains of STAT3 and PRTN3 by SPR method. f SDS/PAGE analysis of hPR3-generated fragments of STAT3. At different times, the incubation mixtures were separated on a 10% SDS/PAGE gel denatured/reduced conditions and visualized by silver nitrate staining (n = 3). g The solvent-accessible surface of the hPR3 active site was generated with Yasara (http://www.yasara.org). h Identification of the cleavage sites within the whole STAT3 after incubation with PRTN3 at pH 7.4 and 37 °C. Cleavage products were monitored by MALDI-TOF MS. Arrows indicate the cleavage sites of STAT3 that were identified. The width of the arrows indicates the intensity of the cleavage. Data are the mean ± s.d.; n: biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.