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. Author manuscript; available in PMC: 2016 Jun 23.
Published in final edited form as: Cell Rep. 2015 Jun 11;11(11):1737–1748. doi: 10.1016/j.celrep.2015.05.031

Figure 5. GSNL-1 acts downstream of CED-3 and can be cleaved by CED-3.

Figure 5

(A) gsnl-1 acts in the same pathway with CED genes. Double mutants of gsnl-1 and CED genes have similar degree of SNB-1-GFP elimination defects as single mutants. (B) The F-actin severing function of gsnl-1 is required for synapse elimination induced by activation of the cell death pathway. Overexpression of gsnl-1 can partially suppress ced-3 phenotypes in RME neurons, and deleting the linker region required for severing function abolishes its suppression ability on ced-3(lf). (C-D) CED-3 can cleave GSNL-1. (C) Western blotting analysis shows loss of function in ced-3 suppresses the cleavage of GSNL-1. FLAG antibodies recognize the full length (50kd) and the cleaved (40kd) GSNL-1. No signal was detected in wild type N2 worms without the transgene. Pttx-3∷gfp was used as a co-injection marker for gsnl-1 transgene, and GFP was served as a loading control in western blot. (D) Quantification of cleavage rate of GSNL-1. Five independent experiments were performed, and band intensity was measured using ImageJ. The cleavage rate was calculated by: P40 Intensity/(P50 intensity+ P40 intensity) × 100%. In Figure A and B, experiments were performed at least 3 times, with N ≥80 animals each time. For transgenic animals the results shown here are generated from at least three independent lines. Data is shown as mean ± SD. Student test, ** P < .01, ns: no significant difference.