Figure S1. Verification of ihog Mutation by Protein Immunoblotting Figure S2. Verification of ihog and boi

Transcription

1 Figure S1. Verification of ihog Mutation by Protein Immunoblotting Extracts from S2R+ cells, embryos, and adults were analyzed by immunoprecipitation and immunoblotting with anti-ihog antibody. The Ihog protein is absent in ihog homozygotes. An anti-tubulin immunoblot is included as a loading control. Figure S2. Verification of ihog and boi Alleles Generated by Targeted Recombination (A, B) Primers used for RT-PCR are shown as blue arrows in Figure 5; pgk was used as an internal control. (A) An ihog + transcript (P1+P2, 124 bp) was detected in wild-type and ihog mutant heterozygotes, but not in ihog mutant homozygotes. A hybrid ihogwhite transcript (P1+P3, 153bp) was detected in ihog heterozygous or homozygous mutants, but not in wild-type. (B) A boi + transcript (P5+P6, 160bp) was detected exclusively in wild-type and a larger transcript (P5+P6, 3233 bp) exclusively in boi homozygotes. The absence of the 160 bp wild-type product presumably is due to deletion of the splice acceptor for exon 5 in the boi mutant, thus preventing incorporation of P5 sequences into transcripts. The 3233 bp fragment in the mutant corresponds to an unspliced transcript, absent in the wild-type. With primers P4 and P6, the 1620 bp fragment (P4+P6, 1620 bp) represents a normally spliced boi-rb or boi-rd transcript, whereas the 207 bp product (P4+P6, 207 bp) corresponds to a mutant transcript corresponding to direct splicing from exon 3 to exon 7 (exon 6 within boi-ra begins at an internal initiation site and lacks a splice acceptor). Figure S3. Boi Proteins Encoded by Predicted Transcripts from the Wild-Type and Mutant boi Locus The predicted Boi-RA, Boi-RB and Boi-RD proteins are aligned by sequence. Replacement of exon 4 and part of exon 5 of boi by white results in deletion of the signal sequence (SS, orange) and the four Ig domains (blue) from the coding sequences of the altered Boi proteins in the boi mutant (Boi-RB* and Boi-RD*), making it unlikely that they would be normally produced and secreted; the predicted Boi-RA protein in wild-type and mutant alleles is also unlikely to be secreted.

2 Figure S4. Smo Functions Downstream of Ihog/Boi Wing imaginal discs from boi hemizygous larvae carrying ihog mutant MARCM clones (boi/y; ihog) were immunostained for GFP (green), Ci (red) and Smo (blue). Smo protein levels were reduced in the mutant clone located adjacent to the AP compartment boundary (yellow asterisk), originating from the A compartment (as judged by the expression of Ci). Figure S5. Ihog/Boi Is Not Required for Hh Release Normal Ptc expression (red) adjacent to a large boi; ihog homozygous mutant clone (marked by loss of GFP expression) originating from the P compartment (marked by lack of Ci expression). Figure S6. IhogFn1,2 Complexed with HhN The molecular surface of a crystallographic complex between HhN (green) and Ihog Fn1, 2 (wheat) is shown, with the location of two missense mutations in surface residues of Fn2 (K653E and Q655E) in red. As shown, HhN contacts IhogFn1 (McLellan et al. 06); IhogFn2 is required for interaction with Ptc (see text). Figure S7. Surface Localization of Ptc Is Ihog/Boi Dependent Confocal microscope images showing localization of Ptc proteins in S2R+ cells cotransfected with Ptc and either control dsrna or dsrna against both ihog and boi. Cells were fixed with 4% paraformaldehyde and immunostaining with anti-ptc antibodies was done in the absence of detergent permeabilization to emphasize proteins on the cell surface (same as in Figure 4); some intracellular proteins nevertheless were detected. (A) Ptc was mainly localized in intracellular vesicles with a trace of cell surface expression (arrow; see also Figure 4E) in the presence of control RNAi. (B) Cell-surface expression of Ptc protein is reduced in the presence of dsrna against both ihog and boi. 2

3 Figure S8. Co-expression of Ihog with either Ptc or Ptc1130 Increased the Amount of HhN Bound to Cells Confocal microscope images of S2 cells transfected by Ptc or Ptc1130 with or without Ihog, as indicated. After incubation with HhN conditioned medium for 2 hours at 4 C (to prevent endocytosis), cells were fixed, permeabilized (with 0.1% Triton X-100 in PBS) and immunostained. (A) Ptc was mainly localized in intracellular vesicles with a trace of surface expression, and very little HhN was detected on the surface of Ptc expressing cells. (B) Ptc1130 was mainly localized on the cell surface and more HhN was detected on the surface of cells expressing Ptc1130 as compared to cells expressing full length Ptc (Lu et al., 2006). (C) Co-expression of Ihog dramatically increased the amount of surface localized Ptc protein and significantly more HhN was detected on the cell surface as compared to A. (D) Co-expression of Ihog dramatically increased the amount of HhN bound to cells expressing Ptc1130 as compared to cells expressing Ptc1130 alone (B). Figure S9. Purification and Immobilization of HhN-GluGlu Anti-GluGlu affinity matrix with bound HhN-GluGlu (2 µl) was incubated with sample loading buffer and analyzed by SDS-PAGE (15% gel) and Coommassie Brilliant Blue staining. The migration of molecular mass markers is indicated on the left, and bands corresponding to the antibody chains and HhN-GluGlu on the right. Note the nearly equivalent intensities of bands corresponding to HhN-GluGlu and antibody light chain, which indicates the near-saturation of the anti-gluglu matrix. Figure S10. A Complex of Ihog/Boi, Ptc, and Hh Requires Both Fn1 and Fn2 GluGlu tagged HhN protein was purified using Sepharose beads containing the anti- GluGlu monoclonal antibody and these beads were incubated with detergent-solubilized (1% NP40, 50 mm Tris-HCl at ph 6.8, 150 mm NaCl, 40uM low molecular weight Heparin, 1mM CaCl2, and protease inhibitors) extracts from S2R+ cells expressing Ptc (lane 1), or Ptc together with HA-tagged Ihog variants (lanes 2-4) or Boi variants (lanes 5-7). Precipitation of Ptc by Sepharose HhN-GluGlu beads depended critically on the 3

4 presence of Ihog or Boi (lanes 2 and 5), and was abolished by deletion of either Fn1 or Fn2 domain of Ihog/Boi (lanes 3,4, 6 and 7). In comparison, precipitation of Ihog/Boi with anti-ha beads precipitated significantly less Ptc (compare lanes 9 and 12 with lanes 2 and 5, respectively). Note that, similar to Ihog (Figure 4B and 4C), the Fn1 of Boi is required to interact with HhN (lane 6, middle panel) and Fn2 of Boi is required to interact with Ptc (lane 14, lower panel). Table S1. Primers Used in Figure 1 and Figure S2 4

5 Zheng_FigS1 wt boi ihog/+ ihog wt ihog IP anti-ihog WB anti-ihog 5% Input Lysate WB anti-tubulin S2R+ Cells Fly embryos Adult flies

6 Zheng_FigS2 A wt ihog/+ ihog P1+P2 124bp P1+P3 153bp PGK 267bp B wt boi P5+P6 160bp 3233bp P4+P6 207bp 1620bp PGK 267bp

7 Zheng_FigS3 SS Boi-RA 1 0 Boi-RB 1 MAEIRLVVVLLVLMTTCIYT LQASDSNSSVATTLGVLFERAPESAVAPKGDEVVFECE LNLKPDRLEWRFRRGDSSEPYLYLRPSAGYNVTSGSDGLAWRLRIYVSAQTAGDYQCVAWYGPGALASTPARLALVSIALDV 140 Boi-RD 1 MAEIRLVVVLLVLMTTCIYT LQASDSNSSVATTLGVLFERAPESAVAPKGDEVVFECE LNLKPDRLEWRFRRGDSSEPYLYLRPSAGYNVTSGSDGLAWRLRIYVSAQTAGDYQCVAWYGPGALASTPARLALVSIALDV 140 Boi-RB* 1 0 Boi-RD* 1 0 Ig Boi-RA 1 0 Boi-RB 141 GQGSMGARSSIRWSVAPKNCLLIRCGSVI SNPPAIWSFYRNGKKLPQSELLPGAAGALVLDTVTAKDAGNYSCVATNSITGDELRLPQTIELR VDYTDRTPPYFLQRPPIEYSARPGETVVLECPGVGSPRP RVIWSSPN 280 Boi-RD 141 GQGSMGARSSIRWSVAPKNCLLIRCGSVI SNPPAIWSFYRNGKKLPQSELLPGAAGALVLDTVTAKDAGNYSCVATNSITGDELRLPQTIELR VDYTDRTPPYFLQRPPIEYSARPGETVVLECPGVGSPRP RVIWSSPN 280 Boi-RB* 1 0 Boi-RD* 1 0 Boi-RA 1 0 Boi-RB 281 VVEIYNNRSTILSYGLQITDVKPEDQGSYICMLDNGIAPPIDHTIKLSVLQRPTILR GPAATLTNESNPLLLDCSASGNPQPDI YWLINGEDATKDPEAVVDNRSLQLKRVQKRHAGVVQCFAKNILGETSEGNILRVNP 420 Boi-RD 281 VVEIYNNRSTILSYGLQITDVKPEDQGSYICMLDNGIAPPIDHTIKLSVLQRPTILR GPAATLTNESNPLLLDCSASGNPQPDI YWLINGEDATKDPEAVVDNRSLQLKRVQKRHAGVVQCFAKNILGETSEGNILRVNP 420 Boi-RB* 1 0 Boi-RD* 1 0 Boi-RA 1 MIWSGNARYSNVQN------S SMVPPSRPNVTRLSDESVMLRWSVPQNSGLQITFFKVQYRMLGDGKNRRKNWQTTSDNIPYGNAHGSGPGQGNSHWRARNRDREYEMGNPPKNF 109 Boi-RB 421 LEITGEDDEPLGEVPVWPVHESNTGMGTSSTSGGSKNKSGRRKF KASMVPPSRPNVTRLSDESVMLRWSVPQNSGLQITFFKVQYRMLGDGKNRRKNWQTTSDNIPYGNAHGSGPGQGNSHWRARNRDREYEMGNPPKNF 560 Boi-RD 421 LEITGEDDEPLGEVPVWPVHESNTGMGTSSTSGGSKNKSGRRKF KASMVPPSRPNVTRLSDESVMLRWSVPQNSGLQITFFKVQYRMLGDGKNRRKNWQTTSDNIPYGNAHGSGPGQGNSHWRARNRDREYEMGNPPKNF 560 Boi-RB* 1 MVPPSRPNVTRLSDESVMLRWSVPQNSGLQITFFKVQYRMLGDGKNRRKNWQTTSDNIPYGNAHGSGPGQGNSHWRARNRDREYEMGNPPKNF 93 Boi-RD* 1 MVPPSRPNVTRLSDESVMLRWSVPQNSGLQITFFKVQYRMLGDGKNRRKNWQTTSDNIPYGNAHGSGPGQGNSHWRARNRDREYEMGNPPKNF 93 ********************************************************************************************* Boi-RA 110 TSSVTGLTAGKYYRFRI VAVYSNNDNKEGNTSLKFFLQNGTSKSNLPVPELREVETLSESAVVLHWSLVSSTQNEDIDGYYAYYRPADSAAEYLKATVDGGMS RSFKIDLLRPGTPYEFKLQSFNSDAASEFSAIRQART 249 Boi-RB 561 TSSVTGLTAGKYYRFRI VAVYSNNDNKEGNTSLKFFLQNGTSKSNLPVPELREVETLSESAVVLHWSLVSSTQNEDIDGYYAYYRPADSAAEYLKATVDGGMS RSFKIDLLRPGTPYEFKLQSFNSDAASEFSAIRQART 700 Boi-RD 561 TSSVTGLTAGKYYRFRI VAVYSNNDNKEGNTSLKFFLQNGTSKSNLPVPELREVETLSESAVVLHWSLVSSTQNEDIDGYYAYYRPADSAAEYLKATVDGGMS RSFKIDLLRPGTPYEFKLQSFNSDAASEFSAIRQART 700 Boi-RB* 94 TSSVTGLTAGKYYRFRI VAVYSNNDNKEGNTSLKFFLQNGTSKSNLPVPELREVETLSESAVVLHWSLVSSTQNEDIDGYYAYYRPADSAAEYLKATVDGGMS RSFKIDLLRPGTPYEFKLQSFNSDAASEFSAIRQART 233 Boi-RD* 94 TSSVTGLTAGKYYRFRI VAVYSNNDNKEGNTSLKFFLQNGTSKSNLPVPELREVETLSESAVVLHWSLVSSTQNEDIDGYYAYYRPADSAAEYLKATVDGGMS RSFKIDLLRPGTPYEFKLQSFNSDAASEFSAIRQART 233 ******************************************************************************************************************************************** TM Fn2 Boi-RA 250 RKTHEQP ASSSATPVPANKAVEQHQNSLYP LIAGAAGGGILLLIASLVVCLCLKRRENSQPEDENKPQLEHIQADFVTSAVLGVGGHHKSGDVRRLNGVIPRMNITPNPLAQETASDK Boi-RB 701 RKTHEQP ASSSATPVPANKAVEQHQNSLYP LIAGAAGGGILLLIASLVVCLCLKRRENSQPEDENKPQLEHIQADFVTSAVLGVGGHHKSGDVRRLNGVIPRMNITPNPLAQETASDK Boi-RD 701 RKTHEQP ASSSATPVPANKAVEQHQNSLYP LIAGAAGGGILLLIASLVVCLCLKRRENSQPEDENKPQLEHIQADFVTSAVLGVGGHHKSGDVRRLNGVIPRMNITPNPLAQETASDKSPMFQNRNVMELRFLPPLANGN 840 Boi-RB* 234 RKTHEQP ASSSATPVPANKAVEQHQNSLYP LIAGAAGGGILLLIASLVVCLCLKRRENSQPEDENKPQLEHIQADFVTSAVLGVGGHHKSGDVRRLNGVIPRMNITPNPLAQETASDK Boi-RD* 234 RKTHEQP ASSSATPVPANKAVEQHQNSLYP LIAGAAGGGILLLIASLVVCLCLKRRENSQPEDENKPQLEHIQADFVTSAVLGVGGHHKSGDVRRLNGVIPRMNITPNPLAQETASDKSPMFQNRNVMELRFLPPLANGN 373 ********************************************************************************************************************** Boi-RA NNSGLHQNGLHHAQPYHPPGTPTLMHKRLDYHQQPPPVPPHSAYYQQAPT 417 Boi-RB NNSGLHQNGLHHAQPYHPPGTPTLMHKRLDYHQQPPPVPPHSAYYQQAPT 868 Boi-RD 841 GNGNCMARDLALERSNAEEQQDEDGHEDEKGLPAVASSSCETLEACDAAIKLNLPQKHDQEQLDAPNAVLEAPSKPLPPLPKKPANGAGQNNSGLHQNGLHHAQPYHPPGTPTLMHKRLDYHQQPPPVPPHSAYYQQAPT 980 Boi-RB* NNSGLHQNGLHHAQPYHPPGTPTLMHKRLDYHQQPPPVPPHSAYYQQAPT 401 Boi-RD* 374 GNGNCMARDLALERSNAEEQQDEDGHEDEKGLPAVASSSCETLEACDAAIKLNLPQKHDQEQLDAPNAVLEAPSKPLPPLPKKPANGAGQNNSGLHQNGLHHAQPYHPPGTPTLMHKRLDYHQQPPPVPPHSAYYQQAPT 513 ************************************************** Boi-RA 418 HQPSPMMERSVRGHQQPGGFHMEEGTPTPTRIPSLRRQRRTSGNQQVHNSHSNLNIGLSHHNNNNNNLPPHHQHLHHHPGHPGGLVGIPIVPGSPRVQRSPMPSRAMIKRTRLGSHTDNISSGSLNSIEV 547 Boi-RB 869 HQPSPMMERSVRGHQQPGGFHMEEGTPTPTRIPSLRRQRRTSGNQQVHNSHSNLNIGLSHHNNNNNNLPPHHQHLHHHPGHPGGLVGIPIVPGSPRVQRSPMPSRAMIKRTRLGSHTDNISSGSLNSIEV 998 Boi-RD 981 HQPSPMMERSVRGHQQPGGFHMEEGTPTPTRIPSLRRQRRTSGNQQVHNSHSNLNIGLSHHNNNNNNLPPHHQHLHHHPGHPGGLVGIPIVPGSPRVQRSPMPSRAMIKRTRLGSHTDNISSGSLNSIEV 1110 Boi-RB* 402 HQPSPMMERSVRGHQQPGGFHMEEGTPTPTRIPSLRRQRRTSGNQQVHNSHSNLNIGLSHHNNNNNNLPPHHQHLHHHPGHPGGLVGIPIVPGSPRVQRSPMPSRAMIKRTRLGSHTDNISSGSLNSIEV 531 Boi-RD* 514 HQPSPMMERSVRGHQQPGGFHMEEGTPTPTRIPSLRRQRRTSGNQQVHNSHSNLNIGLSHHNNNNNNLPPHHQHLHHHPGHPGGLVGIPIVPGSPRVQRSPMPSRAMIKRTRLGSHTDNISSGSLNSIEV 643 ********************************************************************************************************************************** Fn1

8 Zheng_FigS4 * * * Smo GFP B Ci

9 Zheng_FigS5 Overlay anti-gfp anti-ptc anti-ci * *

10 Zheng_FigS6 Ihog Fn1 Q655E K653E! HhN Ihog Fn2

11 Zheng_FigS7 A Ptc + boi RNAi + ihog RNAi Ptc + yfp RNAi B

12 Zheng_FigS8 Ptc HhN Overlay Ptc A Ptc1130 B Ptc1130+Ihog Ptc+Ihog C D

13 Zheng_FigS9 75kD 50kD Mouse anti-gluglu IgG (H) 25kD Mouse anti-gluglu IgG (L) 20kD HhN-GluGlu

14 Zheng_FigS10 anti-gluglu (HhN) anti-ha (Ihog or Boi) anti-hhn anti-ha anti-ptc GFP + Ptc Ihog + Ptc Ihog Fn1 + Ptc Ihog Fn2 + Ptc Boi + Ptc Boi Fn1 + Ptc Boi Fn2 + Ptc GFP + Ptc Ihog + Ptc Ihog Fn1 + Ptc Ihog Fn2 + Ptc Boi + Ptc Boi Fn1 + Ptc Boi Fn2 + Ptc Precipitation Immobilized Protein

15 Table S1. Primers Used in Figure 1 and Figure S2 Primers Location Sequence P1 exon 1 of ihog ATAAGTGATGCGCGATAGCC P2 exon 2 (deleted region) of ihog AGGATGTGAGCAGCGTCATT P3 white AGGTCGACTCTAGAGGATCA P4 exon 3 of boi AGCAGATACGCCTGGAAAGA P5 exon 5 (not deleted region) of boi GCGGCAGACGAAAGTTTAAG P6 exon 7 of boi ACATGCGATACTGCACCTTG