Supplementary materials
Table 1 Oligonucleotide primers for PCR amplification of UMOD
Exon
Sequence (5'3')
forward
GAGTACAAGGGGTAAAGTCCCAGCAAAA
reverse
AAGTGCGATAGGAGGATTGAGATCACCT
forward
AGTTCCCTGGAGAATGAGGGAAGGATCT
reverse
CAGGCGTAGCCCTCCCCGTACT
forward
ATGTGTCAATGTGGTGGGCAGCTACTT
reverse
GGTCACAGGGACAGACAGACAATCAATA
forward
GTACTGCACAGGTCAGCCGGAGTCT
reverse
GTGGATCTTCTGTTTTCACTCAGGTTGG
forward
CTCAAGGCTATGCTGAGCACTTCCAGAT
reverse
AAACTGAGGCACAGCCAGGCTAAATAAC
forward
CCTACTCAGATTCTCATGCCCCTTTCTC
reverse
AGTCTAGATTTGAACCTGGGCTCCAGAA
forward
CAACAGTTGGTGGGTTCCACTCTTATTG
reverse
AAAGATGCAATTTTCCTCCATCCAAGTC
forward
TATCTTATCACATGGGCTGCGATCATTT
reverse
TCTGGTAAAAGAGGGAAACAGGGAAGAA
forward
TATCTAACAAATGGCAGAGCTGGGACCT
reverse
CCAATGAATGGGTGTATGAATGGGAATA
forward
TGGCTGCGAAGTGAAAAGTAGATTGAAG
reverse
ACTCAGGTTCTCTGAGCCACTCTCCTTA
forward
GTGTCCTCTTCTGATTGGTCAGCCTAGA
reverse
GTCACAAGTCCCATTTTGAGAAAAAGCA
2
3a
3b
4
5
6
7
8
9
10
11
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Fig.S1 The novel mutation of uromodulin C112Y found in the FJHN family.
(A) The family pedigree of FJHN patients. Family members with symptoms are indicated by a blackened
within the symbol. † = death, N.E. = not examined.
(B) Partial sequence electropherogram of UMOD gene of the propositus (upper panel) and schematic
diagram of uromodulin structure (lower panel). The C112Y mutation located in the Ca-binding site of EGF-
like domain encoded in the exon 3. UMOD gene consists of 11 exons. Exon 2-11 are coding, and exon 1 is
UTR. EGF = epidermal growth factor-like domain, cb = calcium binding motif, CCS = consensus cleavage
site, GPI = glycosyl-phospatidylinositol-anchoring site, D8C = the domain of eight cysteines, ZP-N = zona
pellucida-N subdomains, ZP-C = zona pellucida-C subdomains, IHP = internal hydrophobic patch, EHP =
external hydrophobic patch, Y = glycosylation sites.
Fig.S2 Effects of MG132 on protein solubility.
Soluble and insoluble fractions of cell lysates 48 hours after transfection with or without treatment of
MG132 (5μM) were subjected to anti-uromodulin Western blotting.
Fig. S3 Effect of lactacystine on the intracellular protein levels of WT and C112Y mutant.
Representative immunoblots of mature, immature forms and secreted form of WT and C112Y mutant with
or without treatment of lactacystine (25 μM). Cells were co-transfected with the WT or C112Y mutant and
2
EGFP plasmid. Cell lysates were prepared 48 hours after co-transfection and were subjected to Western
blot with the indicated antibodies.
Fig. S4 Effects of topiroxostat on the proteins level of intracellular and secreted WT and C112Y
Representative immunoblots of mature, immature forms and secreted form of WT and C112Y mutant with
or without treatment of topiroxostat (30 μM). Cells were co-transfected with the WT or C112Y mutant
and EGFP plasmid. Cell lysates were prepared 48 hours after co-transfection and were subjected to Western
blot with the indicated antibodies. The condition media were prepared 48 hours after co-transfection,
concentrated and were subjected to Western blot with the indicated antibodies.
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