Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein
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1 Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein content relative to GAPDH in two independent experiments. ARPE-19 cells were transfected with a vector containing the human AdipoR1 empty vector, AdipoR1 shrna, and a non-specific shrna (negative control). Mouse skeletal muscle lysate was used as a positive control. The first lane displays the marker showing bands at 52 KDa and 31 KDa. (b, c) Densitometric measurement of western blots from three independent experiments showing an increase of 81% in AdipoR1 protein content in ORF expressing cells (b) and decrease of 38.1% in AdipoR1 silenced cells (c). Responses are expressed relative to controls. AdipoR1 was standardized using GAPDH. shrna, short hairpin RNA; ORF, open reading frame.
2 Supplementary Figure 2. ELOVL4 is not down-regulated in AdipoR1 -/- retinas. (a) Western blot demonstrating no significant difference in ELOVL4 concentration between AdipoR1 +/+ and -/- retinas. (b) Immunohistolocalization of ELOVL4 in an AdipoR1 +/+ retina. Anti-ELOVL4 label (green) appears within the ONL and inner segments of photoreceptors. (c) Immunohistolocalization of ELOVL4 in an AdipoR1 -/- retina. Anti-ELOVL4 label (green) also appears within the ONL and inner segments of photoreceptors. ONL, outer nuclear layer. Nuclei appear blue (DAPI labeling). 23- day-old mice were used for both western blot analysis and immunolocalization. ONL, outer nuclear layer. Magnification bar, 50 µm.
3 Supplementary Figure 3. Full fragmentation spectra and molecular structures of -/- VLC-PUFAs. (a) Spectrum of PC 54:8 (m/z = = M(1025.8) + Hac (60) H) shows the fatty acid composition as FA20:4 and FA34:4. m/z of 724 is produced from (M-(FA20:4-H)+2H, m/z 499 from FA34:4 and m/z 303 from FA20:4. (b) Spectrum of PC 54:7 (m/z = = M(1027.8) + Hac (60) H) shows the fatty acid composition as FA20:4 and FA34:3. m/z of 726 is produced from (M-(FA20:4-H)+2H, m/z 501 from FA34:3, m/z 528 from (M-(FA34:3-H)+2H, and m/z 303 from FA20:4. (c) Spectrum of PC 56:9 (m/z = = M(1051.7) + Hac (60) H) shows the fatty acid composition as FA20:4 and FA36:5. m/z of 750 is produced from (M-(FA20:4-H)+2H, m/z 525 from FA36:5, and m/z 303 from FA20:4. (d) Spectrum of PC 56:8 (m/z = = M(1053.7) + Hac (60) H) shows the fatty acid composition as FA20:4 and FA36:4. m/z of 752 is produced from (M-(FA20:4-H)+2H, m/z 527 from FA36:4, and m/z 303 from FA20:4. m/z of 331 can be from FA22:4, and m/z 499 from 36:5. (a) PC54:8 is composed of FA20:4 (arachidonic acid) and FA34:4. (b) PC 54:7 is composed of FA20:4 (arachidonic acid) and FA34:3. (c) PC 56:9 is composed of FA20:4 (arachidonic acid) and FA36:5. (d) PC 56:8 is composed of FA20:4 (arachidonic acid) and FA36:4, or FA22:4 and FA34:4. The molecular structures of these four phospholipids accompany each spectrum.
4 Supplementary Figure 4. Sphingomyelin levels in AdipoR1 +/+ and -/- retinas does not change. (a) Chromatogram of retention times for phosphatidylcholine and sphingomyelin, illustrating excellent separation of species. (b, c) Mass spectra of AdipoR1 +/+ and -/- retinas, demonstrating that sphingomyelin levels are not affected by the lack of AdipoR1.
5 Supplementary Figure 5. Generation of the AdipoR1 -/- mice. (a) Design of the gene trap mutation to produce the AdipoR1 -/- mice, and the RT-PCR analysis of the AdipoR1 +/+ and -/- mice to demonstrate the loss of the transcript in the AdipoR1 -/- animals. (i) Gene trap mutation of the AdipoR1 gene. SA, splice acceptor sequence; βgeo, beta galactosidase-neomycin resistance fusion gene. The dotted line in the wild-type (AdipoR1 +/+ ) locus indicates normal splicing from exon 5 into exon 6, leading to normal transcript expression. The dotted line in the mutated locus indicates splicing from exon 5 into the gene trap vector rather than exon 6, leading to disruption of the normal transcript. (ii) AdipoR1 RT-PCR. Primers C and D are complementary to AdipoR1 exons 5 and 6 flanking the insertion site of the gene trapping vector. (iii) RT-PCR, using primers C and D, shows absence of endogenous message in kidney and spleen of AdipoR1 -/- animals. RT, reverse transcription.targeted disruption of the AdipoR1 gene locus. (b) Targeting strategy used to disrupt the AdipoR1 locus. Homologous recombination (represented by X) between the targeting vector and the AdipoR1 gene results in the replacement of exons 2-4 with the selection cassette. (c) Southern hybridization indicating proper gene targeting in the embryonic stem cell clones.
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doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
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Probe. Hind III Q,!?R'!! /0!!!!D1"?R'! vector. Homologous recombination
Supple-Zhang Page 1 Wild-type locus Targeting construct Targeted allele Exon Exon3 Exon Probe P1 P P3 FRT FRT loxp loxp neo vector amh I Homologous recombination neo P1 P P3 FLPe recombination Q,!?R'!!
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Supplementary Fig. S1. Schematic diagram of minigenome segments.
open reading frame 1565 (segment 5) 47 (-) 3 5 (+) 76 101 125 149 173 197 221 246 287 open reading frame 890 (segment 8) 60 (-) 3 5 (+) 172 Supplementary Fig. S1. Schematic diagram of minigenome segments.
Nature Genetics: doi: /ng.3731
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Supplemental Figure 1
Supplemental Figure 1 A S100A4: SFLGKRTDEAAFQKLMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGFPDK Overlap: SF G DE KLM LD N D VDFQEY VFL I M N FF G PD S100A2: SFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDR
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Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1 Supp. Fig. 2 CD4 + CD44 low CD4
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Target Protein Antibody name Product number Manufacturer Species Epitope Dilution Aggrecan Anti-aggrecan AB1031 EMD Millipore Corp Rabbit
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Figures S1-S5, Figure Legends, Table S1 List of primers used in the study
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