Rtaq premix

In order to identify polymorphisms in the promoter region of UBXN1 gene, four
pairs of primers (Table 1, A–D) were designed to amplify the transcription
regulatory region from ca. $-$ 2322 to ca. $-$ 176 bp (the A of the initiation
codon ATG was denoted as $+$ 1) for subsequent direct sequencing (Invitrogen,
Shanghai, China). The single nucleotide polymorphism (SNP) was genotyped by Sanger sequencing. A 207 bp
fragment harboring the SNP ca. $-$ 379T  $\mathit{>}$  G was amplified with primer E
(Table 1, E) in 10  $\mathrm{\mu }$ L reaction mixture which contained 100 ng DNA
templates, 5  $\mathrm{\mu }$ L rTaq Premix (Takara, Dalian, China), 0.5  $\mathrm{\mu }$ L of
forward and reverse primers (10  $\mathrm{\mu }$ mol) (Invitrogen, Shanghai, China), and
3  $\mathrm{\mu }$ L ddH ${}_{\mathrm{2}}$ O. The cycling protocol was 5 min at 94  ${}^{\circ }$ C, 35 cycles of 94  ${}^{\circ }$ C for 30 s, 68  ${}^{\circ }$ C annealing for 30 s and
72  ${}^{\circ }$ C for 30 s, with a final extension at 72  ${}^{\circ }$ C for 7 min. Then PCR products were sent to GENEWIZ (Suzhou, China) for Sanger
sequencing.

The 357 bp fragment which harbors GG and TT genotypes from ca. $-$ 677 to ca. $-$ 320
was amplified with primer H. The 20  $\mathrm{\mu }$ L amplification system
consisted of 200 ng DNA templates, 10  $\mathrm{\mu }$ L rTaq Premix (Takara, Dalian,
China), 1  $\mathrm{\mu }$ L of forward and reverse primers (10  $\mathrm{\mu }$ mol) (Invitrogen,
Shanghai, China), and 7  $\mathrm{\mu }$ L ddH ${}_{\mathrm{2}}$ O. The cycling protocol was 5 min at
94  ${}^{\circ }$ C, 35 cycles of 94  ${}^{\circ }$ C for 30 s, 64  ${}^{\circ }$ C
annealing for 30 s, and 72  ${}^{\circ }$ C for 30 s, with a final extension at
72  ${}^{\circ }$ C for 7 min. The forward primer was added NheI restriction
enzyme cutting site, and reverse primer was added HindIII restriction
enzyme cutting site. The two genotype fragments were then cloned into the
pGL3-basic vector (Promega, USA) separately. Twenty-four hours before
transfection, 293T cells were seeded in each well of 12-well plates.
The 293T cells were co-transfected with the constructed reporter plasmid and pRL-TK plasmid (Promega, USA). The transfection system consisted of 1  $\mathrm{\mu }$ g TT
or GG genotype reporter plasmid, 0.05  $\mathrm{\mu }$ g pRL-TK plasmid, 100  $\mathrm{\mu }$ L
OPTI-MEM, and 3  $\mathrm{\mu }$ L Lipofectamine^® 2000 Reagent. Experiments were performed in biological triplicate.
Twenty-four hours after transfection, cells were lysed in passive lysis
buffer (Promega, USA). Firefly luciferase activity and Renilla luciferase
activity were measured according to the manufacturer's protocol in three
independent experiments (Promega, USA).