Protein inhibitor

Cells were collected and washed with PBS. Thereafter, proteins were extracted using RIPA lysis buffer containing protein inhibitor (Solarbio). Afterward, 300 μL extracts were added with 50% Protein A/G beads (Upstate Biotechnology, Lake Placid, NY, USA) for 1 h-incubation at 4°C for pre-clearing. After centrifugation to remove the beads, the supernatant was obtained and incubated overnight with antibody: anti-hnRNPU (1 μg/mL, A300-690A, Thermo Fisher Scientific) or normal rabbit IgG (1:1000, ab172730, Abcam) (negative control) at 4°C, followed by 2 h-incubation with 50% Protein A/G beads at 4°C. Later, the protein-antibody complexes were rinsed with RIPA buffer for 5 min × 5 times, followed by resuspension of protein-antibody complex beads in buffer containing SDS. Immunoprecipitated proteins were boiled at 95–100°C for 3–5 min and the supernatant was collected by centrifugation. The hnRNPU and actin protein levels were then analyzed by WB assay [52 (link),53 (link)].

Protein extract: protein inhibitor (Solarbio) + 20 mM Tris-HCl solution (pH7.5, Solarbio). Adherent cells in flasks were digested and prepared into cell suspension. The suspension was mixed with 1 mL extract, and the mixture was pipetted repeatedly until the complete lysis of cells. The extract was centrifuged in a precooled centrifuge at 4°C for 20 min at 1.6×10⁴ ×g. Protein concentration in the supernatant was determined by bicinchoninic acid (BCA), and the protein was separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, the separated protein was transferred to a nitrocellulose (NC) membrane and left for 1 h at room temperature (sealed with 5% skim milk-phosphate buffer saline (PBS)). Then testing protein and anti-beta Actin antibody (Abcam) were added, and placed overnight at 4°C. The NC membrane was washed three times with PBS solution, then added with goat anti-rabbit secondary antibody (horseradish peroxidase (HRP) conjugate, Abcam), and left to stand for 1 h at room temperature. Finally, the NC membrane was washed with PBS and visualized with enhanced chemiluminescence (ECL) solution. β-actin was taken as the internal reference protein, and the relative expression of the protein = gray value of test band/gray value of the β-actin band.