Real-time quantitative PCR, proteasome degradation pathway and
ER-stress analysis
For RT-PCR experiments, total RNA was extracted from APRE19 cells using
an RNA kit (Thermo GeneJET RNA Purification Kit, Thermo scientific), and
then reverse-transcribed into cDNAs (RevertAidTM First
Strand cDNA Synthesis Kit, Thermo Scientific). The real-time primer
pairs upstream of the mutation site were 5’-ACAAGCTAGGGGACAATGCC-3 ’and
5’-AACCAGCCGCACATAGTCTC-3’, while the downstream mutation site primers
were 5’-ACAAAGAGCTGCTGGGGATC-3 ’and 5’-CTAGCGGCCTCATGAGATGG-3’.
After transfection of the wild-type and mutant recombinant vectors for
48 hours, a total protein extraction was performed and the differences
in the expression of ER-stress markers detected. Similarly, 48 hours
after transfection of wild-type and mutant recombinant vectors,
c.569C>G (p.S190*) transfected cells were pretreated with 0
μM, 25 μM, and 50 μM of the MG132 proteasome inhibitor (Sigma-Aldrich)
for 24 hours. Normal APRE19 cells served as the control, total proteins
were extracted and the change in ARR4 content measured. Target proteins
were detected according to the following procedures. First, the
harvested cells were washed with PBS and lysed with a 63 mM Tris HCl,
10% glycerol, and 2% SDS lysis buffer. The extracted proteins were
subjected to 10% SDS-PAGE electrophoresis and then transferred to a
polyvinylidene fluoride membrane (Millipore, Billerica, MA). Second, the
membrane was washed with Tris buffer, blocked using 5% skim milk powder
in 0.1% Tween-20, and then incubated with the primary and secondary
antibodies according to the manufacturer’s instruction. Finally, an
enhanced chemiluminescence blotting detection kit (GE Healthcare,
Buckinghamshire, UK) was used to detect protein bands.