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1 Supplementary Materials for Dok-7 ctivates the Muscle Receptor Kinase MuSK and Shapes Synapse Formation kane Inoue, Kiyoko Setoguchi, Yosuke Matsubara, Kumiko Okada, Nozomi Sato, Yoichiro Iwakura, Osamu Higuchi, Yuji Yamanashi* *To whom correspondence should be addressed. Published 4 February 009, Sci. Signal., ra7 (009) DOI: /scisignal This PDF file includes: Fig. S1. The phs-dok-7-egfp transgene is overexpressed in skeletal muscle. Fig. S. The number of myofibers was comparable in and Dok-7 mice. Fig. S3. ChR clusters formed only in Dok-7 / myotubes that express Dok-7- EGFP. Fig. S4. Transcripts for endogenous Dok-7 were distributed uniformly throughout the muscle at E18.5. Fig. S5. The Myc epitope appropriately increased the molecular mass of phosphorylated. Fig. S6. Dok-7 activated. Fig. S7. MuSK was not activated by a Dok-7 deletion mutant lacking the PH domain or the Dok-family protein Dok-1. Fig. S8. The phs-dok-7-myc transgene is overexpressed in skeletal muscle.
2 fig. S1 C HS promoter Dok-7 EGFP p muscle heart brain liver EGFP (Transcript) EGFP (Protein) IP: Dok-7 I: GFP D E Dok-7 E14.5 E18.5 E14.5 E18.5 IP: Dok-7 I: GFP I: actin E18.5 IP: Dok-7 I: Dok-7 fig. S1. The phs-dok-7-egfp transgene is overexpressed in skeletal muscle. () The 5 regulatory region of the HS gene (HS promoter) was fused to the EGFP-tagged Dok-7 cdn to generate the phs-dok-7-egfp transgene. () The phs-dok-7-egfp transgene is expressed uniformly throughout the muscle. Whole mounts of diaphragm muscles from wild-type () or Dok-7 transgenic (Dok-7 ) embryos were processed for in situ hybridization using a probe specific to EGFP (left panel). Fluorescence emitted from Dok-7-EGFP was also visualized (right panel). Scale bars, µm. (C) The phs-dok-7-egfp transgene is expressed specifically in skeletal muscle. Tissue lysates prepared from or Dok-7 () mice were subjected to immunoprecipitation (IP) followed by immunoblotting (I). (D and E) The expression level of the phs-dok-7-egfp transgene increases from E14.5 to E18.5 of embryogenesis (D) and is higher than that of the endogenous Dok-7 gene of controls (E). Limb muscle lysates were prepared from or Dok-7 () embryos and subjected to IP and/or I. rrowheads indicate positions of Dok-7-EGFP and arrows indicate positions of actin (D) or Dok-7 (E). The bands observed between Dok-7-EGFP and Dok-7 are most likely degradation products of Dok-7-EGFP (E). Numbers to the left (D) or the right (E) of the immunoblot panels indicate molecular size markers (in kilodaltons).
3 fig. S Relative amount Dok-7 fig. S3 ChR Dok-7-EGFP grin 16 h Number of myofibers fig. S. The number of myofibers was comparable in and Dok-7 mice. Cross-sections of diaphragm muscles from or Dok-7 embryos at E18.5 were stained with hematoxylin and eosin (Fig. 1, P and Q), and the number of myofibers in a defined area of the diaphragm muscle was quantified. The mean value from myofibers was arbitrarily defined as 1. Error bars indicate S.D.M. (n = 3 embryos for each genotype). fig. S3. ChR clusters formed only in Dok-7 -/- myotubes that express Dok-7-EGFP. Dok-7 -/- myotubes were transfected with Dok-7-EGFP expression plasmids, treated with agrin, and stained with fluorescently-labeled α-bungarotoxin to visualize acetylcholine receptors (ChR). Fluorescence emitted from Dok-7-EGFP was also visualized. Scale bars, 0 µm.
4 fig. S4 E14.5 E18.5 Dok-7 fig. S4. Transcripts for endogenous Dok-7 were distributed uniformly throughout the muscle at E18.5. Diaphragm muscles were prepared from embryos and subjected to in situ hybridization using an RN probe specific to Dok-7. Scale bars, µm. fig. S5 1 -Myc C -Myc Dok-7-His -Myc PY--Myc PY- fig. S5. The Myc-epitope appropriately increased the molecular mass of phosphorylated. The bacterially expressed cytoplasmic region of MuSK () or Myc-tagged (-Myc) was purified, separated (10% SDS-PGE), and visualized with Coomassie rilliant lue (C) staining. rrowheads indicate the positions of each purified protein (left panel). Purified recombinant proteins as indicated were incubated for h in phosphorylation buffer and subjected to immunoblotting (I) (right panel). PY, phosphotyrosine. Numbers to the left of the immunoblot panels indicate molecular size markers (in kilodaltons).
5 fig. S6 TP 0 - Dok-7-His ( o C) (h) PY-Dok-7-His PY- 1 -K Dok-7-His -K PY-Dok-7-His PY- C fig. S6. Dok-7 activated. () Purified in the presence or absence of Dok-7-His were incubated for h at 30 C or 4 C in phosphorylation buffer, either complete or lacking TP, and subjected to immunoblotting (I). () acterially expressed or a kinase-inactive mutant (-K) was purified, separated (10% SDS-PGE), and visualized with Coomassie rilliant lue (C) staining. n arrowhead indicates the position of the purified proteins (left panel). The purified recombinant proteins were incubated for h in phosphorylation buffer and subjected to I (right panel). PY, phosphotyrosine. Numbers to the left of the immunoblot panels indicate molecular size markers (in kilodaltons).
6 fig. S7 1 Dok-7-His Dok-7 N-His Dok-7-His Dok-7 N-His (min) PY- C 1 Dok-1-His Dok-7-His Dok-7-His Dok-1-His (min) PY- C fig. S7. MuSK was not activated by a Dok-7 deletion mutant lacking the PH domain or the Dok-family protein Dok-1. acterially expressed, His-tagged Dok-7, Dok-7- N (N-terminal deletion mutant which lacks the PH domain), or Dok-1 was purified, separated (10% SDS-PGE), and visualized with Coomassie rilliant lue (C) staining. and ). rrowheads indicate the positions of each purified protein (left panel; The purified recombinant proteins indicated were incubated in phosphorylation buffer for the indicated period and subjected to immunoblotting (I) (right panel; and ). PY, phosphotyrosine. Numbers to the left of the immunoblot panels indicate molecular size markers (in kilodaltons).
7 fig. S8 E18.5 Myc HS promoter Dok-7 p IP : Dok-7 I : Dok-7 fig. S8. The phs-dok-7-myc transgene is overexpressed in skeletal muscle. () The 5 regulatory region of the HS gene (HS promoter) was fused to the Myc-tagged Dok-7 cdn to generate the phs-dok-7-myc transgene. () The expression level of the phs-dok-7-myc transgene is higher than that of the endogenous Dok-7 gene of wild-type () controls. Limb muscle lysates were prepared from or Dok-7 embryos and subjected to immunoprecipitation (IP), followed by immunoblotting (I). The input from Dok-7 embryos is a quarter of that from controls. n arrowhead indicates the position of Dok-7-Myc and arrows indicate the positions of Dok-7. The number to the left of the immunoblot panel indicates a molecular size marker (in kilodaltons).
Supplementary Figure 1. α-synuclein is truncated in PD and LBD brains. Nature Structural & Molecular Biology: doi: /nsmb.
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Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table.
Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table. Name Sequence (5-3 ) Application Flag-u ggactacaaggacgacgatgac Shared upstream primer for all the amplifications of
Figure S1. Figure S2. Figure S3 HB Anti-FSP27 (COOH-terminal peptide) Ab. Anti-GST-FSP27(45-127) Ab.
/ 36B4 mrna ratio Figure S1 * 2. 1.6 1.2.8 *.4 control TNFα BRL49653 Figure S2 Su bw AT p iw Anti- (COOH-terminal peptide) Ab Blot : Anti-GST-(45-127) Ab β-actin Figure S3 HB2 HW AT BA T Figure S4 A TAG
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GFP CCD2 GFP IP:GFP
D1 D2 1 75 95 148 178 492 GFP CCD1 CCD2 CCD2 GFP D1 D2 GFP D1 D2 Beclin 1 IB:GFP IP:GFP Supplementary Figure 1: Mapping domains required for binding to HEK293T cells are transfected with EGFP-tagged mutant
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DOI: 10.1038/ncb3363 Supplementary Figure 1 Several WNTs bind to the extracellular domains of PKD1. (a) HEK293T cells were co-transfected with indicated plasmids. Flag-tagged proteins were immunoprecipiated
Supplementary Fig. 1
a FL (1-2266) NL (1-1190) CL (1191-2266) HA-ICE1: - HA-ICE1: - - - FLAG-ICE2: + + + + FLAG-ELL: + + + + + + IP: anti-ha FLAG-ICE2 HA-ICE1-FL HA-ICE1-NL HA-ICE1-CL FLAG-ICE2 b IP: anti-ha FL (1-2266) NL
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Supplementary Figure 1 Pol structure-function analysis (a) Inactivating polymerase and helicase mutations do not alter the stability of Pol. Flag epitopes were introduced using CRISPR/Cas9 gene targeting
Supplement Figure S1:
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