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Supplemental Figure 1: Anti-GDF15 antibody blocks binding of GDF15 to hGFRAL. Anti-GDF15 antibody (mAb26, a neutralizing monoclonal antibody to wtGDF15) blocks binding of GDF15 to human GFRAL in a dose-dependent manner by SPR-based blocking assay. Nature Medicine: doi:10.1038/nm.4394

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Page 1: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 1: Anti-GDF15 antibody blocks binding of GDF15 to

hGFRAL. Anti-GDF15 antibody (mAb26, a neutralizing monoclonal antibody to wtGDF15)

blocks binding of GDF15 to human GFRAL in a dose-dependent manner by SPR-based

blocking assay.

Nature Medicine: doi:10.1038/nm.4394

Page 2: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 2: Human GFRAL interacts with GDF15 but not GDNF family

ligands. Surface plasmon resonance of the binding of GDF15 and GDNF family ligands

(GDNF, NRTN, ARTN and PSPN) to immobilized human GFRAL and a non-relevant protein

as negative control.

Nature Medicine: doi:10.1038/nm.4394

Page 3: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 3: RET binds to the GFRAL/GDF15 complex as analysed by

flow cytometry. Human GFRAL(ECD)-HEK cells were shown to binding to GDF15 and to

RET but only in the presence of GDF15.

Nature Medicine: doi:10.1038/nm.4394

Page 4: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 4: Tissue Expression of GFRAL and RET mRNA in lean rats.

Tissues (n=4) were collected from lean male SD rat and gene expression was measured

by qPCR. ND: not detectable. 1/4, 2/4 and 3/4 indicates the number of tissues with

measurable expression mRNA levels.

Nature Medicine: doi:10.1038/nm.4394

Page 5: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 5: Original images of representative uncropped

immunoblots with molecular weight standards. pRET (Y905), pRET (Y1062), RET,

beta-Actin

pRET(Y905) 100kDa 140kDa

pRET(Y1062) 100kDa 140kDa

RET 100kDa 140kDa

35kDa β-actin

MARKER

MARKER

MARKER

MARKER

Nature Medicine: doi:10.1038/nm.4394

Page 6: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 6: Original images of representative uncropped

immunoblots with molecular weight standards. pERK (T202/Y204), ERK, pAKT

(S473), AKT

pERK (T202/Y204) 40kDa

55kDa

40kDa 55kDa ERK

pAKT(S473) 70kDa

70kDa AKT

MARKER

MARKER

MARKER

MARKER

Nature Medicine: doi:10.1038/nm.4394

Page 7: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Figure 7: Original images of representative uncropped

immunoblots with molecular weight standards. pAMPK (T172), pSTAT3 (Y705),

pSMAD (s463/465), GAPDH

pAMPK(Thr172) 70kDa

pSTAT3 (Y705)

130 kDa

100 kDa

70 kDa

70 kDa

55 kDa

40 kDa

pSmad

55 kDa

40 kDa

35 kDa

GAPDH

MARKER

MARKER

MARKER

MARKER

Nature Medicine: doi:10.1038/nm.4394

Page 8: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Table 1. Screened molecules related to the TGFβ receptor family.

Binding of these 140 molecules were tested against GDF15 alone, GFRAL alone and

complex of GFRAL and GDF15 by flow cytometry. None of them showed detectable

binding.

NO. Gene CCDS# NO. Gene CCDS# NO. Gene CCDS#

1 ACVR1 CCDS2206.1 48 FGFR2 CCDS44486.1 95 NCAM1 CCDS73385.1 2 ACVR1B CCDS8816.1 49 FGFR2 CCDS7620.2 96 NCAM1 CCDS73386.1 3 ACVR1C CCDS2205.1 50 FGFR2 CCDS44485.1 97 NCAM1 CCDS73387.1 4 ACVR2A CCDS33301.1 51 FGFR2 CCDS53584.1 98 NEO1 CCDS53957.1 5 ACVR2B CCDS2679.1 52 FGFR2 CCDS44488.1 99 NEO1 CCDS58378.1 6 ACVRL1 CCDS31804.1 53 FGFR3 CCDS54706.1 100 NOMO1 CCDS10556.1 7 ALK CCDS33172.1 54 FGFR3 CCDS3353.1 101 NOTCH1 CCDS43905.1 8 AMHR2 CCDS8858.1 55 FGFR4 CCDS4410.1 102 NTRK1 CCDS30891.1 9 APLP1 CCDS32997.1 56 FGFRL1 CCDS3344.1 103 NTRK1 CCDS1161.1 10 APP CCDS33523.1 57 FLT4 CCDS4457.1 104 NTRK1 CCDS30890.1 11 APP CCDS13576.1 58 FURIN CCDS10364.1 105 NTRK2 CCDS6671.1 12 APP CCDS13577.1 59 FXYD6 CCDS8387.1 106 NTRK3 CCDS32322.1 13 APP CCDS56213.1 60 GFRA1 CCDS7593.1 107 NTRK3 CCDS58399.1 14 APP CCDS56211.1 61 GFRA1 CCDS44481.1 108 PCDHA4 CCDS54916.1 15 BAMBI CCDS7162.1 62 GFRA3 CCDS4201.1 109 PCSK5 CCDS55320.1 16 BMPR1A CCDS7378.1 63 GFRA3 CCDS4201.1 110 PIK3IP1 CCDS13893.1 17 BMPR1B CCDS58919.1 64 GFRA4 CCDS13055.1 111 PMEPA1 CCDS13463.1 18 BMPR1B CCDS3642.1 65 GFRA4 CCDS13056.1 112 RRBP1 CCDS13128.1 19 BMPR2 CCDS33361.1 66 GREB1 CCDS42655.1 113 SDC1 CCDS1697.1 20 BOC CCDS2971.1 67 HFE CCDS4580.1 114 SDC2 CCDS6272.1 21 BTC CCDS3566.1 68 HFE CCDS4582.1 115 SDC3 CCDS30661.1 22 CACNA2D3 CCDS54598.1 69 HFE CCDS4581.1 116 SDC3 CCDS30661.1 23 CD34 CCDS31011.1 70 HFE CCDS54975.1 117 SDC4 CCDS13350.1 24 CD79A CCDS12589.1 71 HFE2 CCDS910.1 118 SEL1L CCDS9876.1 25 CD79A CCDS46088.1 72 HFE2 CCDS910.1 119 SEL1L CCDS58333.1 26 CD99 CCDS14119.1 73 HLA-B CCDS34394.1 120 SORT1 CCDS55618.1 27 CD99 CCDS48071.1 74 IL17RA CCDS13739.1 121 ST14 CCDS8487.1 28 CDH1 CCDS10869.1 75 IL1RL1 CCDS2057.1 122 TDGF1 CCDS2742.1 29 CDH11 CCDS10803.1 76 JAG2 CCDS9998.1 123 TFR2 CCDS34707.1 30 CDH2 CCDS11891.1 77 JAG2 CCDS9999.1 124 TGFBR1 CCDS6738.1 31 CDON CCDS8468.1 78 KITLG CCDS31868.1 125 TGFBR2 CCDS2648.1 32 CHST3 CCDS7312.1 79 KITLG CCDS31867.1 126 TGFBR2 CCDS33727.1 33 DLL1 CCDS5313.1 80 KLRB1 CCDS8601.1 127 TGFBR3 CCDS30770.1 34 EDAR CCDS2081.1 81 LFNG CCDS34586.1 128 TGFBR3 CCDS55614.1 35 EGF CCDS54794.1 82 LFNG CCDS34587.1 129 TLR2 CCDS3784.1 36 EGF CCDS3689.1 83 LNPEP CCDS43346.1 130 TMCO2 CCDS30684.1 37 EGFR CCDS5514.1 84 LRCH4 CCDS34706.1 131 TNF CCDS4702.1 38 ENG CCDS6880.1 85 LRP5 CCDS8181.1 132 TNFRSF10D CCDS6038.1 39 EPHA2 CCDS169.1 86 LRRC32 CCDS8245.1 133 TNFRSF10D CCDS6038.1 40 ERBB2 CCDS32642.1 87 MCAM CCDS31690.1 134 TNFRSF14 CCDS44046.1 41 F11R CCDS1213.1 88 MET CCDS47689.1 135 TNFRSF18 CCDS10.1 42 FGF3 CCDS8195.1 89 MET CCDS43636.1 136 TNFSF11 CCDS9384.1 43 FGFR1 CCDS43730.1 90 MFRP CCDS8421.1 137 TNFSF14 CCDS12171.1 44 FGFR1 CCDS43731.1 91 MLANA CCDS6466.1 138 TNMD CCDS14469.1 45 FGFR1 CCDS55222.1 92 MME CCDS3172.1 139 VASN CCDS10514.1 46 FGFR1 CCDS43732.1 93 MMP14 CCDS9577.1 140 ZP2 CCDS10596.1 47 FGFR2 CCDS31298.1 94 NCAM1 CCDS73384.1

Nature Medicine: doi:10.1038/nm.4394

Page 9: Supplemental Figure 1: Anti-GDF15 antibody blocks binding ......Supplemental Table 1. Screened molecules related to the TGFβ receptor family. Binding of these 140 molecules were tested

Supplemental Table 2. Metabolic phenotype of GFRALKO female mice

WT

Day

WT

Night

GFRAL KO

Day

GFRAL KO

Night

VO2 [ml/ hour] 71.8±1.7 88.5±1.6*** 69.2±3.1 82.1±3.4***

RER [VCO2/VO2] 0.86±0.01 0.98±0.01*** 0.96±0.01 0.85±0.01***

EE [kcal/h/kg] 14.3±0.4 18.1±0.2*** 14.7±0.5 17.7±0.6***

Activity [beam

breaks]

346±32 999±107*** 361±35 855±81***

VO2: oxygen uptake, VCO2: carbon dioxide release, RER: respiratory exchange ratio, EE:

energy expenditure. Statistical analysis performed using two-ANOVA with repeated

measures and Holm Sidak post-doc analysis, n=8. ***, p<0.001 significant difference

between light and dark period. Data are expressed as mean±SEM of average light and

dark periods over a 5 day period.

Nature Medicine: doi:10.1038/nm.4394


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