Hnrnpu

Nucleoprotein extracts were treated with RNase (Solabio, China) and incubated with antibodies for hnRNPK (1:50) or IgG for 24 h on a rotating wheel. Thereafter, the Sepharose-conjugated protein-A/G beads were added and incubated at 4°C for another 24 h on a rotating wheel. After extensive rinsing with cold PBS, the beads were boiled, and the precipitated proteins were separated via SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature and subsequently incubated with primary and secondary antibodies, and protein bands were detected via enhanced chemiluminescence. The primary antibodies used were as follows: hnRNPK (Proteintech, #11426-1-AP, 1:1000), IgG (CST, #2729, 1:1000), hnRNPA1 (Proteintech, #11176-1-AP, 1:5000), hnRNPR (Proteintech, #15018-1-AP, 1:1000), hnRNPU (Proteintech, #16365-1-AP, 1:5000), Histone H3 (Abclonal, #A2348, 1:1000) and β-Actin (Proteintech, #20536-1-AP, 1:2000).

The primary antibodies were at the dilution of 1:1000, including HNRNPU (16365–1-AP, ProteinTech Group, China), HA (51064–2-AP, ProteinTech Group, China), c-Myc (s1826, Clontech, Japan), Alix (2171S, Cell Signaling Technology, USA), CD9 (13174S, Cell Signaling Technology, USA), Ubiquitin (10201–2-AP, ProteinTech Group, China), β-actin (4970S, Cell Signaling Technology, USA). The goat anti-rabbit IgG/Alexa fluor 647 (BS-0295-G, Bioss, China) was at the 1:200 dilution. The second antibodies goat anti-rabbit IgG(H + L) HRP (FMS-Rb01, Fcmacs) and goat anti-mouse IgG (H + L) HRP (S0002, Affinity) were in 5000 diluted concentrations. GW4869 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).