Figure 3. Molecular properties of lipoprotein-associated and lipoprotein-free Hh proteins.

(A) Size of lipoprotein-associated and lipoprotein-free Shh secreted from HeLa cells grown in the presence or absence of FBS. Supernatants were fractionated by gel filtration chromatography on a Superdex 200 column, and fractions analyzed by WB. Molecular weight standards are indicated. Note that large mammalian lipoproteins run in the void volume of these columns. (B) Hemolymph Hh levels in Drosophila larvae ectopically expressing Hh in the fat body, with or without concomitant Lpp RNAi, analyzed by WB. (C) Density of hemolymph Hh produced in the fat body of control or Lpp RNAi larvae, analyzed by Optiprep density gradient centrifugation and WB. (D) Size of Lpp-associated and Lpp-free hemolymph Hh ectopically produced in the fat body of control or Lpp RNAi larvae, analyzed by gel filtration chromatography as described in (A). (E) Cholesterol modification status of low- and high-density forms of Drosophila Hh. Supernatant from Hh-producing S2 cells grown in the presence of FBS and ³H-cholesterol was fractionated by KBr density gradient centrifugation, and subsequently analyzed by both WB and in-gel fluorography. (F) Hydrophobicity of low- and high-density forms of Hh. Supernatants from Hh-producing S2 cells grown in the presence of serum were fractionated by KBr density gradient centrifugation; fractions 1 and 8 were analyzed by Triton X-114 phase separation and WB. (G–I) Hydrophobicity of Shh and Hh produced in the presence or absence of lipoproteins, assessed by Triton X-114 phase separation and WB. (G) Lysates and media from HeLa cells expressing Shh and grown in the presence or absence of FBS. (H) Hemolymph from control and Lpp RNAi larvae. (I) Hemolymph from control and Lpp RNAi larvae ectopically producing Hh in the fat body. Note that aqueous phase Hh/Shh has a lower electrophoretic mobility than detergent phase Hh/Shh.