Igg mouse

Subconfluent HDLECs were fixed in 4%  (v/v) paraformaldehyde in PBS, permeabilized in 0.3% Triton and blocked in 2% (w/v) BSA/PBS 0.05% Tween for 1 h. Primary antibodies [TRBP (Abcam, ab42018) 2.5 μg/ml, S6K2 (Abnova, B02P), S6K1 (Abcam, ab119252), 2.5 μg/ml, IgG Mouse (Cell Signaling) 12.5 μg/ml, IgG Rabbit (Cell Signaling) 2.5 μg/ml and Dicer (Abcam) 20 μg/ml)] were incubated overnight at 4°C in blocking buffer. Washes were performed in blocking buffer, followed by a final wash in 10% blocking buffer/PBS. PLA was performed using Duolink, as per the manufacturer's instructions (Olink Biosciences, Sigma, DUO92102). Cells were stained for F-actin with Alexafluor 488 Phalloidin (Life Technologies, A12379) 1:40 (v/v) in blocking buffer for 20 min, and then mounted in DAPI-containing mounting media, for nuclear staining (Sigma, DUO82040). Images were taken using a Zeiss LSM 510 inverted confocal microscope and the number of PLA events per cell in 100 cells per condition was counted.

The BxPc‐3 cells were fixed by the blocking solution following the manufacture’s protocol (Duolink in situ fluorescence; Sigma). Then, the primary antibodies MIB1 (sc‐393551; Santa Cruz Biotechnology, Beijing, China; 1 : 200 dilution) and ST7 (ab122459; Abcam; 1 : 100 dilution), or IgG (Rabbit) (3900; Cell Signaling Technology; 1 : 5000 dilution) and IgG (Mouse) (53484; Cell Signaling Technology; 1 : 5000 dilution) were applied to incubated with the cells for 2 h at 37 °C. Then, 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. Thirty minutes later, cells were incubated with amplification–polymerase solution for 100 min. The Duolink In Situ Mounting Medium with DAPI was added to cells to take photos under confocal microscope.

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)].