Figure 4: Secreted TyrRS is cleaved by matrix metalloproteinase 2 during cell competition.

(a) Western blot with protein extracts of larvae expressing TyrRS-HA-tagged protein incubated with the anti-HA antibody, full-length TyrRS-HA is detected and a smaller fragment product of proteolytic cleavage (TyrRS51-HA) are detected. (b) Western blot of the third instar larvae haemolymph 48 h after cell competition clones were induced. Genotype ywF; tub>dmyc>gal4, UAS-LacZ (control), UAS-RNAi Mmp1, UAS-RNAi Mmp2, UAS-Mmp2DN. Lip-Lipophorin, TyrRS-full-length TyrRS, TyrRS 52, 46 and 41 kDa are cleaved fragments of TyrRS, *Unspecific band, 17-kDa fragments corresponds to EMAP and/or MiniTyr molecular weight. Lipophorin was used as a loading control. (c) Quantification of total secreted TyrRS/total TyrRS protein normalized by the loading control (Lipophorin) in haemolymph of the third instar larvae 48 ACI; experiments represent the average of three independent experiments. * is statistically significant, T-test P<0.05; s.d. is represented. (d–i) Haemocyte staining (Nimrod, red) in wing imaginal discs with clones of wt loser cells (green, GFP) under cell competition 48 h after heat shock (ACI). (d,e) Control loser cells expressing LacZ, (f,g) loser cells expressing Mmp1 RNAi and (h,i) loser cells expressing Mmp2 RNAi. (j,k) Haemocyte staining (Nimrod, red) in wing imaginal discs with clones of cells (green, GFP) expressing secreted TyrRS (secTyrRS) and Mmp2 RNAi 48 h after heat shock (ACI). Nuclei are marked in blue (DAPI). (l) Number of haemocytes per disc quantification (n=10 discs). * is statistically significant, T-test P<0.05; s.d. is represented. Scale bar, 50 μm. DAPI, 4,6-diamidino-2-phenylindole; NS, not significant.