Supplemental Figure 1: Anti-GDF15 antibody blocks binding of GDF15 to hgfral.
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1 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.
2 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.
3 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.
4 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.
5 pret(y905) 140kDa 100kDa pret(y1062) 140kDa 100kDa RET 140kDa 100kDa β-actin 35kDa Supplemental Figure 5: Original images of representative uncropped immunoblots with molecular weight standards. pret (Y905), pret (Y1062), RET, beta-actin
6 perk (T202/Y204) ERK 55kDa 40kDa 55kDa 40kDa pakt(s473) 70kDa AKT 70kDa Supplemental Figure 6: Original images of representative uncropped immunoblots with molecular weight standards. perk (T202/Y204), ERK, pakt (S473), AKT
7 pampk(thr172) 70kDa 130 kda 100 kda pstat3 (Y705) 70 kda 70 kda psmad 55 kda 40 kda GAPDH 55 kda 40 kda 35 kda Supplemental Figure 7: Original images of representative uncropped immunoblots with molecular weight standards. pampk (T172), pstat3 (Y705), psmad (s463/465), GAPDH
8 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 CCDS FGFR2 CCDS NCAM1 CCDS ACVR1B CCDS FGFR2 CCDS NCAM1 CCDS ACVR1C CCDS FGFR2 CCDS NCAM1 CCDS ACVR2A CCDS FGFR2 CCDS NEO1 CCDS ACVR2B CCDS FGFR2 CCDS NEO1 CCDS ACVRL1 CCDS FGFR3 CCDS NOMO1 CCDS ALK CCDS FGFR3 CCDS NOTCH1 CCDS AMHR2 CCDS FGFR4 CCDS NTRK1 CCDS APLP1 CCDS FGFRL1 CCDS NTRK1 CCDS APP CCDS FLT4 CCDS NTRK1 CCDS APP CCDS FURIN CCDS NTRK2 CCDS APP CCDS FXYD6 CCDS NTRK3 CCDS APP CCDS GFRA1 CCDS NTRK3 CCDS APP CCDS GFRA1 CCDS PCDHA4 CCDS BAMBI CCDS GFRA3 CCDS PCSK5 CCDS BMPR1A CCDS GFRA3 CCDS PIK3IP1 CCDS BMPR1B CCDS GFRA4 CCDS PMEPA1 CCDS BMPR1B CCDS GFRA4 CCDS RRBP1 CCDS BMPR2 CCDS GREB1 CCDS SDC1 CCDS BOC CCDS HFE CCDS SDC2 CCDS BTC CCDS HFE CCDS SDC3 CCDS CACNA2D3 CCDS HFE CCDS SDC3 CCDS CD34 CCDS HFE CCDS SDC4 CCDS CD79A CCDS HFE2 CCDS SEL1L CCDS CD79A CCDS HFE2 CCDS SEL1L CCDS CD99 CCDS HLA-B CCDS SORT1 CCDS CD99 CCDS IL17RA CCDS ST14 CCDS CDH1 CCDS IL1RL1 CCDS TDGF1 CCDS CDH11 CCDS JAG2 CCDS TFR2 CCDS CDH2 CCDS JAG2 CCDS TGFBR1 CCDS CDON CCDS KITLG CCDS TGFBR2 CCDS CHST3 CCDS KITLG CCDS TGFBR2 CCDS DLL1 CCDS KLRB1 CCDS TGFBR3 CCDS EDAR CCDS LFNG CCDS TGFBR3 CCDS EGF CCDS LFNG CCDS TLR2 CCDS EGF CCDS LNPEP CCDS TMCO2 CCDS EGFR CCDS LRCH4 CCDS TNF CCDS ENG CCDS LRP5 CCDS TNFRSF10D CCDS EPHA2 CCDS LRRC32 CCDS TNFRSF10D CCDS ERBB2 CCDS MCAM CCDS TNFRSF14 CCDS F11R CCDS MET CCDS TNFRSF18 CCDS FGF3 CCDS MET CCDS TNFSF11 CCDS FGFR1 CCDS MFRP CCDS TNFSF14 CCDS FGFR1 CCDS MLANA CCDS TNMD CCDS FGFR1 CCDS MME CCDS VASN CCDS FGFR1 CCDS MMP14 CCDS ZP2 CCDS FGFR2 CCDS NCAM1 CCDS
9 Supplemental Table 2. Metabolic phenotype of GFRALKO female mice WT Day WT Night GFRAL KO Day GFRAL KO Night VO2 [ml/ hour] 71.8± ±1.6*** 69.2± ±3.4*** RER [VCO2/VO2] 0.86± ±0.01*** 0.96± ±0.01*** EE [kcal/h/kg] 14.3± ±0.2*** 14.7± ±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.
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