Anti fus

The interaction between BACH2 and FUS was examined in vivo using a Pierce Co‐Immunoprecipitation (Co‐IP) Kit (Thermo Fisher Scientific), according to the manufacturer's protocols. Coupling resin was incubated at 4 °C overnight with the indicated amounts of antibody. The antibody‐coupling resin complexes were then used to precipitate the cell lysates. Anti‐BACH2 (Cell Signaling Technology) and anti‐FUS (ProteinTech) were used to detect the precipitate. For in vitro binding assays, GSH‐agarose beads (Thermo Fisher Scientific) were used to purify the GST or GST‐BACH2 fusion bait protein, and His‐tag purification resin beads (Beyotime Biotechnology) were used to purify the His‐FUS fusion protein. GST protein or GST‐BACH2 fusion protein, which was combined with GSH‐agarose beads, was incubated with His‐FUS fusion protein for 6 h at 4 °C. The resulting bead‒protein‒protein complex was precipitated. Proteins isolated using elution buffer were detected by western blotting using anti‐GST (ProteinTech) and anti‐FUS (ProteinTech).

The total cell protein was extracted using lysis buffer (P0013; Beyotime Biosciences, Shanghai, China). We added a mixture of protease and phosphatase inhibitors (B14002 and B15002; Biotool, Shanghai, China), four times the volume of the cell pellet, to the cell lysate. The total protein (35 μg) was separated via SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD-Millipore, Billerica, MA, USA). The membrane was blocked with 5% skimmed milk (232,100; BD Biosciences, Franklin Lakes, NJ, USA) for at least 2 h and then incubated with the appropriate primary antibody overnight at 4 °C. Peroxidase-conjugated secondary antibody was added and incubated at 37 °C for 1 h. The following primary antibodies were used: Anti-ACAT1 (16215-AP-1, 1:1000), anti-P62/SQSTM1 (66184-1-lg, 1:1000), anti-NRF2 (16396-1-AP, 1:1000), anti-lamin B1 (66095-1-lg, 1:1000), anti-FUS (60160-1-lg, 1:1000), and anti-GAPDH (60004-1-Ig, 1:1000) from ProteinTech Group, and anti-LC3B (3868 s, 1:1000) from Cell Signaling Technology (Denver, MA, USA). The total protein was visualized using an enhanced chemiluminescence method (34,080; Thermo Fisher Scientific, Waltham, MA, USA). Image J software (National Institutes of Health, Bethesda, MD, USA) was used to evaluate each band quantitatively and Prism 5.0 software (GraphPad, La Jolla, CA, USA) was used to compare the signal intensity.

Body cavity based lymphoma-1 (BCBL-1) [51 (link)], BCBL-1 TREx-RTA (gift from Jae Jung, USC) and BJAB RRV cells [19 (link)] were maintained in RPMI supplemented with 1% L-glutamine and 20% FBS. Human embryonic kidney 293T (HEK293T) cells (ATCC CRL-3216) were maintained in DMEM supplemented with 1% L-glutamine and 10% FBS. BCBL-1 cells were induced with 600 μM valproic acid, BCBL-1 TREx-RTA were induced with 1.5 μg/mL doxycycline, BJAB RRV cells were induced with 100 or 500 nM TSA, where indicated. For transfection of BCBL-1 and BCBL-1 TREx-RTA cells, 10 million cells were pelleted and washed once with media lacking serum. Either 2 nmoles of RNaseH targeting oligonucleotide or 15 μg of plasmid DNA were electroporated into 10 million cells in a 0.4 cm cuvette at 975 μF/210 mV. HEK293T cells were transfected with Mirus TransIT-293 reagent according to the manufacturer’s directions. Antibodies used are 1:1000 anti-FUS (Proteintech Group, 11570-1-AP), 1:1000 anti-Histone H4 (Upstate Cell Signaling Solutions, 07–108), 1:2000 anti-GAPDH (Sigma G8795), 1:500 anti-KSHV K8.1 (Advanced Biochemicals Incorporated) and 1:1000 anti-KSHV ORF6 (gift from G. Hayward at The Johns Hopkins University). Cells were treated with 100 μg/mL cycloheximide for the indicated times.