Ab1424

HEK293T cells were transfected with the pCAGGS plasmids encoding HA-tagged VP35 and NP using the PEI reagent according to the manufacturer’s instructions. At 36 h after transfection, the cell lysates were prepared with cold lysate buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% glycerol, and 0.05% NP-40) containing EDTA-free protease inhibitors (Roche). The cell lysates were subsequently mixed with EZview Red Anti-HA Affinity Gel beads (Sigma) and incubated at 4 °C overnight with gentle rocking. After washing with lysate buffer, precipitated proteins were subjected to SDS-PAGE and Western blot analyses with a monoclonal anti-HA antibody (Abcam, ab1424) diluted at 1:5000 and rabbit antiserum to EBOV NP (FS0169) [25 (link)] diluted at 1:2000. The bound antibodies were visualized using Immobilon Western (Millipore). Band intensities were quantified using Amersham Imager 600 Analysis Software.

The following antibodies were used for BrdU, western blot, and co‐IP assay: anti‐BrdU (#ab1893; Abcam), anti‐β‐actin (#sc47778; Santacruz), anti‐JNK1 + JNK2 + JNK3 (phospho‐T183 + T183 + T221) (#ab124956; Abcam), anti‐JNK1 + JNK2 + JNK3 (#ab179461; Abcam), anti‐MED8 (#PA5‐118854; Thermo), anti‐HBRNPA1 (#11176‐1‐AP; proteintech) and anti‐HA (#ab1424; Abcam).

HT29 cells were lysed and centrifuged. The protein content was measured using the BCA kit (Beyotime Institute of Biotechnology, Jiangsu, China), and the concentration was calculated. SDS-PAGE electrophoresis was performed and the proteins were transferred to the nitrocellulose membrane. The membrane was then stained with a 2% Ponceau dye solution. After washing, the membrane was blocked. Then, primary antibodies TRIM29 (ab108627, 1 : 5000, Abcam), MDR1 (ab170904, 1 : 2000, Abcam), P53 (60283-2-Ig, 1 : 5000, Proteintech), HA (ab1424, 1 : 7000, Abcam), Flag (ab205606, 1 : 10,000, Abcam), and β-actin (66009-1-Ig, 1 : 5000, Proteintech) were added, with an incubation time of 2–4 hours at room temperature. After washing, a horseradish peroxidase-labeled secondary antibody HRP-goat anti-mouse IgG (SA00001-1, 1 : 5000, Proteintech) or HRP-goat anti-rabbit IgG (SA00001-2, 1 : 6000, Proteintech) was added, with an incubation time of 1 hour at room temperature. ECL chemiluminescence was utilized for visualization.

HEK 293T cells were seeded on 8-well chamber slides (Watson Co., Ltd) precoated with poly-L-lysine (Cultrex). One day after seeding, the cells were transfected with the plasmids encoding mlEFL35p using TransIT LT1 reagent (Mirus) according to the manufacturer’s instructions. At 24 hours post-transfection, the cells were fixed in 4% (wt/vol) paraformaldehyde for 15 min, and permeabilized by incubation for 5 min in PBS containing 0.4% (vol/vol) Triton X-100. The following procedures were performed at room temperature. The cells were incubated with PBS containing 1% (wt/vol) BSA followed by incubation with the anti-HA antibody (abcam, ab1424, 1:500) for 60 min. After washing with PBST, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, A11001) for 30 min in the dark. Nuclei were stained using 1 μg/ml 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular Probes) for 10 min in the dark. Images were acquired with a 63 × oil objective lens on a Zeiss LSM700 inverted microscope and ZEN 2009 software (Carl Zeiss).

For co-immunoprecipitation (co-IP) analysis, TbISWI, NLP, RCCP, and FYRP were tagged at the C terminus with either a triple Myc epitope using the pMoTAG43M vector (59 (link)) or with a triple HA epitope using the pMoTAG4H vector (54 (link), 55 (link), 59 (link)) in PF and BF cell lines. Cell extracts were prepared as for the TAP tagging protocol (63 (link)), except that 0.1% Nonidet P-40 was added while extracting protein. Sepharose CL-4B columns (GE Healthcare) were prepared with ice-cold IP buffer (150 mm sucrose, 20 mml-glutamic acid, 20 mm HEPES-KOH (pH 7.7), 3 mm MgCl₂, 1 mm DTT, 150 mm KCl, 0.1% Nonidet P-40) and incubated with either monoclonal anti-HA (ab1424, Abcam) or anti-Myc (M5546, Sigma) antibodies or no antibody for 2 h at 4 °C. Crude extract (100 μl) was added to the columns with the immobilized antibodies and incubated for 2 h at 4 °C. Washes were carried out with ice-cold wash buffer (20 mm HEPES-KOH, pH 7.7, 3 mm MgCl_2, 150 mm KCl, 0.1% Nonidet P-40). Purified proteins were eluted into boiling SDS-PAGE loading buffer, boiled for 5 min, and centrifuged at 1000 × g for 7 min. The supernatant was removed, and 15 μl was loaded onto either 8 or 10% polyacrylamide gels.

Each cell lysate was mixed with 2 × sample buffer (Bio Rad) and incubated at 65°C for 15 min. Expressed proteins were separated in sodium dodecyl sulfate (SDS)-polyacrylamide gels (SuperSep Ace 5–20%, Wako) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck). PBS containing 3% (wt/vol) skim milk (Becton Dickinson) and PBS containing 0.05% (vol/vol) Tween 20 (PBST) were used as blocking and wash buffers, respectively. PVDF membranes were incubated with an anti-HA monoclonal antibody (abcam, ab1424, 1:5000), anti-β actin monoclonal antibody (abcam, ab6276, 1:5000), or VP24-specific mouse antiserum for 60 min, washed with PBST, and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, 115-035-062, 1:10000) for 60 min. After washing with PBST, the bound antibodies were visualized with Immobilon Western (Millipore).

293 T cells transfected with overexpressing LMO2-HA were lysed in a lysate containing NP-40 (P0013F, Beyotime, China) and Protein inhibitor. After centrifugating, the supernatant was collected, a part of it was added to 5× protein loading buffer, and then the protein was denatured by heating at 100 °C and retained as input. The other two parts of the supernatant were mixed with protein A/G beads (B23202, Bimake, China) and HA antibody (ab1424, Abcam, USA) or IgG Antibodies (3420, CST, USA) respectively, and rotated overnight at 4 °C. Then, samples were washed three times, lysed in lysis buffer, resuspended in 1× protein loading buffer, denatured by heat, and the supernatant was taken for WB detection or mass spectrometry analysis. Specific immunoprecipitation was performed in NB4 cells with LDB1 antibody and the proteins in the complexes were detected by WB.