Figure 4. Imaginal discs produce Hh-N*.

(A) Immunofluorescence (IF) of wing imaginal discs of MTP/Lpp co-RNAi larvae, stained for Hh, Lpp, and Ptc. A denotes the anterior compartment, P the posterior compartment; yellow lines indicate the compartment boundary. Scale bar = 20 µm. (B) Experimental scheme for the purification of Hh-N* from imaginal discs. Samples for experiments shown in (D–F) were prepared in 100 mM Na₂CO₃, pH 9. (C) WB showing the levels of Hh recovered from imaginal discs with PBS or 100 mM Na₂CO₃, pH 9 after 16,000 g (S1) and 100,000 g (S2) centrifugation steps. Sample amount in each lane corresponds approximately to the imaginal discs of two third instar larvae. (D) Density of Hh in 100,000 g supernatant of imaginal discs dissociated in pH 9 buffer, analyzed by KBr density gradient centrifugation and WB. Hh is present mainly in high-density fractions; in some experiments, minor amounts of Hh can also be detected in low-density fractions. (E) Hydrophobicity of low-density and high-density Hh recovered from imaginal discs, analyzed by Triton X-114 phase separation and WB. (F) Size of high-density Hh recovered from imaginal discs, analyzed by gel filtration chromatography and WB.