Figure 5: Hedgehog protein is associated to EVs with features of exosomes.

(a) A representative western blotting of supernatants (SN1500 and SN45) and EVs obtained from 5 ml of media from mock- (control) or Hh-GFP-transfected Cl8 cells. Similar volume of supernatants (1/350 of the total) and 1/5th of the EVs were applied to the gel. Antibodies against indicated proteins were used as described in Methods. Antibody against Hh showed that Hh is found in EVs, although the Hh-associated to EVs represents less than 10% of the total secreted forms (n=3). (b) EVs from Hh-GFP-transfected cells were fractionated by continuous sucrose density gradient and equal amounts (1/5th) of the resulting fractions analysed by western blotting. Specific antibodies show the presence of Hh and protein markers of exosomes (Ubiquitinated proteins, Hsp70, TSG101 and Rab11) mapping at similar densities. ApoLII was also tested in the gradient, showing a different pattern along the gradient. (c) Western blot analysis of Hh protein in EVs obtained from untransfected Cl8 cells and extract from larvae stage 3 (L3). (d) Luciferase activity in Cl8 cells transfected with ptcΔ136-GL3 Firefly responsible reporter and incubated without (Control) or with EVs obtained from Cl8 cells. Shh recombinant protein was used as positive inducer. Mean±s.d. of three replicates is shown. Significance level (Student’s t-test) **P<0.01.