Flag m2 mouse monoclonal antibody

Worms expressing HA::RNP-6, RBM-39::mKate2 or both were harvested and proteins were extracted using the following standard protocol^{74 (link)}. A solubilization buffer containing 0.5% NP40, 150 mM NaCl and 50 mM Tris, pH 7.4 supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) was used for immunoprecipitation. Flag immunoprecipitation was performed using Dynabeads Protein G (Thermo Fisher Scientific) and FLAG M2 mouse monoclonal antibody (Sigma-Aldrich), following the manufacturers’ protocols. Proteins were eluted from the beads by boiling with Laemmli buffer.

4’, 6-diamidino-2-phenylindole (DAPI) was purchased from Fujifilm Wako. Antibodies were obtained as follows: FLAG M2 mouse monoclonal antibody (1:3000 dilution; F1804, Sigma-Aldrich Japan, Tokyo, Japan), rabbit anti-β-actin antibody (1:3000 dilution; A2066, Sigma-Aldrich Japan), rabbit anti-HNRNPM antibody (1:2000 dilution; HPA024344, Sigma-Aldrich Japan) and mouse anti-SRRM2 antibody (1:2000 dilution; S4045, Sigma-Aldrich Japan), HA (12CA5) mouse monoclonal antibody (1:2000 dilution; GTX16918, GeneTex, Irvine, CA), mouse anti-GAPDH antibody (1:2000 dilution; 016-25523, Fujifilm Wako), sera against THOC1 (1:1000 dilution), THOC2 (1:1000 dilution), THOC5 (1:1000 dilution), ALYREF (1:1000 dilution), CIP29 (1:1000 dilution), UAP56 (1:1000 dilution) and URH49 (1:1000 dilution) have been described previously^{11 (link)}. Anti-RUVBL1 (1:1000 dilution), anti-RUVBL2 (1:1000 dilution), anti-ILF2 (1:1000 dilution), and anti-ILF3 sera (1:1000 dilution) were prepared from immunized Wistar female rats as described previously^{59 (link)} in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Animal Committee in Kyoto University (Animal experiments were approved by the Committee on the Ethics of Animal Experiments of Kyoto University, Experiment permission number: Lif-K17002). The antibodies used in this study are listed in Supplementary Table 4.

The following antibodies were used: PREX1 (D808D) rabbit monoclonal, doublecortin rabbit polyclonal antibody, LLGL1 (D2B5A) rabbit monoclonal antibody; phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] MultiMab rabbit monoclonal antibody mix; TIAM1 rabbit polyclonal antibody; doublecortin rabbit polyclonal antibody, all from Cell Signaling Technology; GAPDH mouse monoclonal from Abcam; Flag M2 mouse monoclonal antibody from Sigma-Aldrich.

The following antibodies were used in this study: HA mouse monoclonal antibody (16B12) (BioLegend, # 901502), FLAG M2 mouse monoclonal antibody (Sigma, # F1804), AR rabbit polyclonal antibody (Millipore, # 06–680) for ChIP, AR N-20 rabbit polyclonal antibody (Santa Cruz, # sc-816) for Western blotting, FKBP5 rabbit polyclonal antibody (Cell Signaling, #8245), H3K4Me1 rabbit polyclonal antibody (EpiCypher, # 13–0040), H3K27Ac mouse monoclonal antibody (EpiCypher, # 13-0045), HIRA(38 (link)), Daxx rabbit polyclonal antibody (39 (link)); BRD4 rabbit polyclonal antibody (Fortis Life Science, # A301-985A100) for Western blotting and mab BL-149-2H5 for ChIP, GR (G-5) mouse monoclonal antibody (Santa Cruz, # sc-393232), H3.3S31Ph rabbit monoclonal antibody (Abcam, # ab92628), Actin (AC-74) mouse monoclonal antibody (Sigma, # A5316).

Worms expressing HA::RNP-6, FLAG-SFA-1 or both were harvested, and proteins were extracted using the Bioruptor method. We used buffer containing 0.5% NP40, 150 mM NaCL and 50 mM Tris pH 7.4 supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) for immunoprecipitation. Flag immunoprecipitation was performed using Dynabeads Protein G (ThermoFisher Scientific) and FLAG M2 mouse monoclonal antibody (Sigma), following manufacturer’s protocols. Proteins were eluted from the beads by boiling with Laemmli buffer.

Chromatin immunoprecipitation (ChIP) experiments were performed as reported previously^{42 (link)}. Cells treated for 1 h in the high-light tolerance assay were harvested by centrifugation at 2000 × g at 4 °C for 2 min and resuspended in KH buffer (20 mM HEPES-KOH, pH7.6, and 80 mM KCl) containing 0.35% formaldehyde (Wako Chemical, Japan). After incubation for 10 min at room temperature, the cross-linking reaction was terminated with 1 M glycine for 5 min. The cross-linked cells were sonicated using the BIORUPTOR® II (Cosmo Bio, Japan) for 10 s (×30) with pauses of 20 s between each sonication cycle and then subjected to ChIP. The target DNA–protein complexes were immunoprecipitated using 100 μL of SURE-beads (Bio-Rad Laboratories) conjugated with 5 μg of FLAG (M2) mouse monoclonal antibody (Sigma-Aldrich) at 4 °C for 1 h. After an overnight cross-link reversion between DNA and protein at 65 °C, the DNA was extracted and purified using the phenol/chloroform/isoamylalcohol purification method. The purified DNA was then subjected to semiquantitative PCR using KOD FX Neo DNA polymerase (TOYOBO) on the SimpliAmp Thermal Cycler (Thermo Fisher Scientific). Real-time qPCR assays were performed using the KOD SYBR® qPCR Mix (TOYOBO) on the Light Cycler 96 system (Roche Diagnostics, Germany). The primers used for PCR analyses are listed in Supplementary Table 7.