Proteinase inhibitors

Crude protein extracts were prepared in the presence of proteinase inhibitors (Sangon, China) and the RiboLock™ Ribonuclease Inhibitor (MBI, CT, USA), and were incubated with an anti-Ar-Larp antibody (HuaAn) at 4 °C overnight, followed by the addition of Protein A Dynabeads (Invitrogen) for 3 h at 4 °C. The beads were washed with extraction buffer twice, and tRNAs were purified using TRIzol reagent (Invitrogen). RNA was loaded onto 8 % urea denaturing polyacrylamide gels prior to hybridization with tRNA oligonucleotide probes at 39 °C.

MCF-7 cells were harvested, and then lysed in RIPA buffer containing PMSF (Solarbio, R0010) and proteinase inhibitors (Sangon Biotech, C600386-0001), and the lysates were clarified by centrifugation at 12,500 rpm for 15 min at 4°C. A BCA Protein Assay Kit (Beyotime Biotechnology, P0012) was used to determine protein concentration. Total cellular protein was separated by SDS-PAGE and electro-transferred onto PVDF membranes (Millipore, ISEQ00010). Then the membranes were blocked with 5% milk powder (Sangon Biotech, A600669-0250) and washed by 0.1% TBS/T for 10 min. Subsequently, the membranes were incubated with primary antibody. The following primary antibodies were used: mouse monoclonal anti-ESR1 (Santa Cruz Biotechnology, sc-8002), rabbit polyclonal anti-DNAJC12 (Proteintech, 12338-1-AP), rabbit polyclonal anti-ERBB4 (Proteintech, 19943-1-AP), or mouse monoclonal anti-β-actin (Beyotime Biotechnology, AF0003) at 4°C overnight. After washing three times in 0.1% TBS/T, membranes were incubated with the secondary HRP-conjugated goat anti-rabbit IgG (H + L) (Beyotime Biotechnology, A0208) or HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime Biotechnology, A0216). Proteins were visualized with an ECL system.