Supplementary Fig. S1
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1 Supplementary Fig. S1 CL IP Unc931Flag IP: αh I: αflg Unc931 CL IP Unc931Flag FL IP: αflg I: αh Shorter fragment Supplementary Fig. S1: Unc931 associates with full length and Cterminal h fragment THP1 cells expressing H alone, H+Unc931FLG or +Unc931FLG. () Left panel indicates western blot of complete lysate (CL) with anti FLG specific b. Right panel indicates immunoprecipitation with anti H b (IP) followed by a western blot with anti FLG b (W). () Left panel indicates western blot of complete lysate (CL) with anti H b. Right panel indicates immunoprecipitation with anti FLG b (IP) followed by a western blot with anti H b (W).
2 Supplementary Fig. S2 LRR14 7Ns (Short) Undefined region 7N (Long) 471 7C(CtermK) LRR15 :...SPSGDSSEVGFCSNRTSVESYEPQVLEQLHYFRYDKYRSCRFKNKESFMSVNESCYKYGQTLDLS... CtermG: YDKYRSCRFKNKESFMSVNESCYKYGQTLDLS... CtermH: YRSCRFKNKESFMSVNESCYKYGQTLDLS... CtermI: SCRFKNKESFMSVNESCYKYGQTLDLS... CtermJ: FKNKESFMSVNESCYKYGQTLDLS... CtermK: ESFMSVNESCYKYGQTLDLS... IL8 [pg/ml] CtermG CtermH CtermI CtermJ 2 C R µg/ml IL8 Positive [%] CtermG CtermH CtermI CtermJ CtermK D R837 15µg/ml +7Ns +7Ns +7Ns +7Ns +7Ns CtermG CtermH CtermI
3 Supplementary Fig. S2: Coexpression of a shorter Nterminal h fragment with the Cterminal h fragment fails to restore functional activity of the Cterminal h fragment () Schematic diagram showing a range of N and C terminal h fragments. The undefined region in the Nterminal h fragment, in which cleavage occurs is shown in blue. green box shows the location of a cysteine thought to play a role in disulfide bond formation between the N and Cterminal fragments. In addition to the longer h Nterminal fragment, corresponding to amino acids 1476 of human Tlr7 (F7N) which was used in the main figures of the manuscript, a shorter Nterminal fragment was made (7Ns), which lacked cysteine at position 475. We also engineered a range of Cterminal h fragments CtermG to CtermJ, in addition to the CtermK used in all the main figures of the manuscript. () ELIS with anti IL8 antibody using tissue culture medium from THP1 cells encoding indicated constructs and stimulated for 24 hrs with or without R837 at concentration of 15µg/ml. Values indicate mean ± S.D. of triplicates. = THP1 cells expressing full length h; CtermG, H, I, J= THP1 cells expressing Cterminal fragments and = THP1 cells expressing. (C) Cellsurface biotinylation of THP1 cells expressing either Htagged full length h () or the indicated Htagged Cterminal h fragments followed by immunoprecipitation using Neutravidin (vidin) and Western lot (W) analysis for Htag. (D) Intracellular FCS staining with anti IL8 antibody of THP1 cells expressing indicated constructs and stimulated for 15 hrs in the presence (black bar) or absence of 15µg/ml R837. Values indicate % of positive cells. = THP1 cells expressing, = THP1 cells expressing full length h; CtermG, CtermH and CtermI = THP1 cells expressing Cterminal fragments; Each cell line was tested for its ability to synthesize IL8 alone () or when the shorter h fragment (7Ns) was coexpressed.
![.. C 8 mut 1 mut 2 THP1 6 4 IL8 [pg/ml] LRR15 mut2 : mut1: mut2: 467 mut1 Undefined region FL αh Shorter fragment 2 αctin R837 THP1 H mut1h mut2h αh D αh + αlamp1 Supplementary Fig.](/docs-images/80/80564103/images/4-1.jpg "S3: Mutating residues in the Nterminal region of renders the receptor nonfunctional. () Schematic diagram showing an alignment of regions of the ectodomain of and mutants.")
4 Supplementary Fig. S3 LRR KQFKRLKVIDLSVNKISPSGDSSEVGFCSNRTSVESYEPQVLEQLHYFRYDKYRSCRFKNKESFMSVNESCYKY......KQFKRLKVIDLSVNKISPSGDSSEVGFCSNRTSVESYEPQVLEQLHYFYDKYSCRFKNKESFMSVNESCYKY......QFKLKVIDLSVNKISPSGDSSEVGFCSNRTSVESYEPQVLEQLHYFRYDKYRSCRFKNKESFMSVNESCYKY... C 8 mut 1 mut 2 THP1 6 4 IL8 [pg/ml] LRR15 mut2 : mut1: mut2: 467 mut1 Undefined region FL αh Shorter fragment 2 αctin R837 THP1 H mut1h mut2h αh D αh + αlamp1 Supplementary Fig. S3: Mutating residues in the Nterminal region of renders the receptor nonfunctional. () Schematic diagram showing an alignment of regions of the ectodomain of and mutants. Underline indicates possible furinlike recognition motifs and the undefined region in which cleavage is thought to occur is depicted in blue. oxes highlight the amino acid residues that were replaced by alanine in the mutants. () THP1 cells expressing indicated constructs were stimulated with R837 (15µg/ml) for 24 hrs, IL8 secretion was then measured by ELIS. (C) ntih western blot of total cell lysate of THP1 cells either expressing or mutants. (D) Confocal showing that both mut1 and mut2 colocalize with Lamp1. Htag staining is shown in red with the late endosome/lysosome marker Lamp1 shown in green. Nuclear counterstain (DPI) is shown in blue. THP1 cells expressing the indicated constructs were PM differentiated for 24 hours. Cells were then fixed, permeabilized, blocked and stained with the indicated antibodies. Images were taken with a Zeiss inverted 78 confocal microscope.
5 Table S1 Primer Primers used for cloning Fwd hh Rev hh h/4 PCR Fwd h/4 PCR Rev mh PCR Rev Sequence: 5 3 (restriction enzyme recognition site) CTTGTCCTGGTCCCGGTGGGG (cli) TGGTGTCGCGCTGCTTTGGCGTGTCT (SalI) GTGTGTCTCCCTGGTCTGTCCCTGTCGTGTGCC TCTTGGTGTGTCG 1 CGCCCCTGTGGTCTTTTCTCTGCGGTGTCGTCC GGGGTCCCT 2 TGGTCTCGGGCTGCTTTGGCGTGTCTGG (XhoI) h Mlu1 Fwd 2 h linker Rev Ist2 lenti Fwd Ist2 lenti Rev Ist2 cl1 Fwd Ist2 SalI Rev Flag Fwd Flag Rev cl1kozak Fwd CtermK FLG Fwd MluI Fwd CTCGCGTGGGCCCCTGGTGTTT (MluI) TCTGGTCCTTTGCCTCCCCTCCGCCCCCCCCCTTCCTC CTCCTCCGCCGTTTCCTTGCCCTGCT 3 CTTGTCCGGCCCGCCC (cli) (amhi) TTGCGGCCGCTTGGCGTGTCTGGCCTCTGGGGTGGTCC CGCTTCTTCTTCGCTTGTGCG 4 (NotI) CTTGTCGGGCCCCTGGTGTT (cli) TGGTGTCGCTTGGCGTGTCTGGCCTC (SalI) GCTCGCGTGCGCGGCTGTGGTTTCCTCTC CTTGTCGTCTCGTCTTTGTGTCTCTGCCCCGGGTTTGG TGTCCCCCTGGTGTTTCCTGTGGCC (cl1) GGCTGTCGCTCGCGTGCGCGGGGCTTCTTTC TGTCTG (cli) CGGCGCGTCCCCTGGTGTTTCCTGTGG (MluI) Nterm Rev (Long) GCCTGTCTTTCTGCCTCCTTGCTC (cli ) Nterm Rev (Short) IRES Fwd IRES Rev Mut1 Fwd Mut1 Rev Mut2 Fwd! Mut2 Rev CCGTGTCTCTGTTGTTTGTTCC(clI) CGGGGTCCTGTCCGCCCCTCTCCCTCCCCCCCCCCTCGTTC TGGCCGG (cli) CCGGGTCCTTGTGGCC (amhi) CTTTTTCGCTTGTGTTGCGCGGTTGCGTTC GTCTGCCTCGCTGCTCTTTCTTGCGTTGT GCTCCTCGCTGTTTGCCTTTGCCTGGTCTG TC GTCTTGCTTTCGTGCTTTTTGTGCCTGCTGGGTT GC 1 old: Part of Fwd primer binding in cdn. 2 old: Part of Rev primer binding in cdn. 3 Italic: cdn sequence coding for linker 4 Italic: cdn sequence coding for H epitope
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