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.