Anti xpo1

GBM cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific, 89900) containing 1% protease inhibitors (Thermo Fisher Scientific, A32953). Proteins concentration was measured using BCA protein assay (Thermo Fisher Scientific, 23225) reagents. A total of 20 μg of proteins were separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride/PVDF membranes (Bio-Rad, 162-0177). Then, after blocked with 5% skim milk for 60 min, membranes were incubated at 4°C overnight with primary antibodies as follow: anti-XPO1 (Cell Signaling Technology, 46249S), anti-p-ATM (Cell Signaling Technology, 5883S), anti-p-CHEK1 (Cell Signaling Technology, 2348S), anti-p-CHEK2 (Cell Signaling Technology, 2661), anti-RAD51 (Cell Signaling Technology, 8875), anti-γH2AX (Cell Signaling Technology, 9718S), anti-SQSTM1 (Cell Signaling Technology, 88588), anti-LC3 (MBL BEIJING BIOTECH CO., LTD, M186-3), anti-RNF168 (Abcam, ab271099). Next the membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7074s). The signal was visualized using Super ECL Plus western blotting Substrate (Thermo Fisher Scientific, 34577) and detected by a ChemiDocXRS system (Bio-Rad).

RIP was performed as described by Chen et al.³⁷ (link) Cells were first fixed, lysed, sonicated and centrifuged. Then, the supernatant was collected and incubated with anti-NXF1 (Proteintech) or anti-XPO1 (Cell Signaling Technology) antibody overnight at 4°C with rotation. A sample incubated with IgG was included as control. The next morning, Dynabeads protein G were added to the antibody conjugated complex and incubated for 2 hours at 4°C with rotation. Afterward, the beads were washed and incubated for 1 hour at 70°C to reverse cross-link. Immunoprecipitated RNA was extracted with RNAeasy Animal Total RNA Isolation Kit (Beyotime), and analyzed by RT-qPCR with the primers listed in Table S2.

Protein was extracted from the cells using RIPA buffer (radioimmunoprecipitation assay buffer) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting (WB) as previously described.²⁰ (link) Nuclear and cytosolic fractions were obtained from U266 and H929 cells using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) per the manufacturer’s protocol. The following antibodies were used for immunoblotting: anti-IKZF1 (Abcam), anti-β-actin (Sigma Aldrich), anti-XPO1, anti-c-MYC, anti-eIF4E, anti-PARP, anti-Bax, and anti-LaminB1 (Cell Signaling Technology).