Proximity Ligation Assay for KPNB1-YBX1 Interaction

The U251MG cells were fixed with the blocking solution following the manufacturer’s protocol (Duolink in situ fluorescence; Sigma). Then, the primary antibodies against KPNB1 (67,597; Proteintech; 1:200 dilution) and YBX1 (20339–1-AP; Proteintech; 1:300 dilution) or IgG (Rabbit) (3900; Cell Signaling Technology; 1:5000 dilution) and IgG (Mouse) (53,484; Cell Signaling Technology; 1:5000 dilution) were added to the cells and incubated for 2 h at 37 °C. Then, the cells were washed with 1× wash buffer and incubated with PLA probe for 1 h at 37 °C. The ligation–ligase was added to cells at 37 °C. After 30 min, the cells were incubated with amplification–polymerase solution for 100 min. The Duolink In Situ Mounting Medium with DAPI was added to cells, and images were taken under a confocal microscope. PLA and DAPI signals were counted under a fluorescence microscope, and a high-resolution intercellular visualization was performed using a confocal microscope (Leica Microsystems, Wetzlar, Germany). Detailed operation is referred to this article [47 (link)].