Rnase inhibitor

The pSP64 polyA vector was purchased from Promega (USA). The GFP ORF was cloned from the vector pEGFP-N1 and inserted into the pSP64 polyA vector by HindIII to obtain the SP64-GFP plasmid. The primers used to clone GFP were as follows: antisense: 5′-GATCCACCGGTCGCCACCA-3′; sense: 5′-GTACAGCTCGTCCATGCCGAGAGT-3′. The plasmid SP64-GFP was separately digested with SacI or EcoRI to generate two different types of linear transcriptional templates. Two types of GFP RNA, without or with a 30-nt poly (A) tail, were obtained in a cell-free in vitro transcription system containing NTP mix, RNase inhibitor (Transgene, China), and SP64 RNA polymerase (NEB, USA). DNA templates were digested by RNase-free DNase, and then RNA transcripts were purified using phenol chloroform.

Cells were lysed with lysis buffer (50 mM Tris–HCl, pH 7.0, 0.5% NP-40, protease inhibitor cocktail (Promega) and 2.5% RNase inhibitor (Transgene)) and incubated on ice for 10 min followed by brief sonication [48 (link)]. Cell lysate was clarified by centrigation at 15,000 rpm for 5 min at 4 °C. Phosphorylation reaction buffer contains 10 mM MgCl₂, 50 μM ATP, 1 mM DTT and 50% cell lysate. 20 μM of mEGFP-YBX1 and 30 μM of PLK1 were added to the reaction buffer and incubated at 37 °C for 30 min to allow PLK1 to phosphorylate YBX1. The phase saperation assay was performed by adding 20 μM of mCherry-APE1 and 10 μM of mEGFP-YBX1 from the phosphorylation reaction to cell lysate. The reaction was kept at RT for 30 min before imaging.