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Fig. 1
Generation of N-CISSOR probe.
(a) N-CISSOR, type-I zebrafish NRG1?1 (zNRG1I?1) constructed with an extracellular mCherry and an intracellular GFP, was expressed under the control of a 5× UAS sequence, which was driven by Gal4. (b) N-CISSOR was designed to monitor intramolecular NRG1 cleavage, which resulted in physical separation of mCherry and GFP. Ectodomain-shedding activities were assessed as relative values of the mCherry/GFP ratios. (c) Cellular localization of N-CISSOR expressed in HEK293T cells compared with those of immunodetected HA-mNRG1I?1 and unlabelled zNRG1I?1. Scale bar: 20??m. (d,e) Western blot analysis of N-CISSOR dynamics. Released (1st panel) and full-length (2nd panel) N-CISSOR fragments were detected using anti-mCherry and anti-GFP antibodies, respectively. ?-actin was detected as an internal control (3rd panel). An asterisk shows the extra bands produced by the intramolecular cleavage of mCherry during the denaturation procedure. (d) Metalloprotease-dependent N-CISSOR cleavage induced by PMA treatment for 20?min. Pre- and co-treatment with GM6001 for 30?min inhibited the PMA-induced release of ectodomain fragments. (e) Time-dependent N-CISSOR release induced by PMA treatment. The supernatants of N-CISSOR-expressing cells were harvested at the indicated time points after PMA or DMSO treatment. (f) Analysis of N-CISSOR bioactivity. Differentiated C2C12 myotubes were treated with conditioned medium from N-CISSOR-expressing cells or control cells. Recombinant NRG1 and its vehicle (BSA) were used as positive and negative controls, respectively. pErbB3 (185?kDa), pAkt (60?kDa), and ?-actin (60?kDa) were detected in the lysates of C2C12 myotubes. (g) A merged image of GFP and mCherry in a N-CISSOR-expressing cell. Intracellular mCherry dots are indicated by arrows. Scale bar: 10??m.