Anti flag m2 antibody

The Gem and Ca_Vβ co-immunoprecipitation experiments were performed as described previously (Despang et al. 2020 ). In brief, tsA cells were harvested 48–72 h after transfection in ice cold PBS and lysed in lysis buffer (50-mM Tris; 100-mM NaCl; 10-mM EDTA; 0.4% TritonX-100; 0.4% NP-40; pH: 7.5 with HCl). Protease inhibitor cocktail tablets (Sigma-Aldrich) were added immediately before lysing cells. Cell lysates were incubated for 1h at 4°C with rotation and afterwards for 30 min on ice. Cell lysates were then centrifuged for 15 min to remove cell debris. Lysate of 400μl of was incubated with 50μl (500μg)-Pierce™ Protein A/G Magnetic Beads (Thermo Scientific) and 5μl-anti-FLAG-Antibody M2 (Sigma-Aldrich) or 10-μl anti-HA 3F10 (Roche) at 4°C overnight. Beads were washed two times for 15–30 min in lysis buffer, and then proteins were eluted in 2x Laemmli buffer, incubated at 50°C for 10 min and frozen at −20°C. Elutions were separated using SDS-PAGE, followed by immunoblotting with anti-HA antibody (1:1000, Covance) and anti-FLAG antibody M2 (1:1000, Sigma-Aldrich).

For ChIP experiments, cells were cross-linked with formaldehyde before chromatin was extracted, sonicated, and incubated with primary antibodies Rabbit Monoclonal anti-Histone H3 (acetyl K27) (Abcam, Cambridge, UK) (#ab4729, 1:500); Mouse monoclonal ANTI-FLAG® M2 antibody (Merck Millipore, Burlington, MA) (#F1804, 1:100); Rabbit Polyclonal anti-BRD4 (Bethyl, Montgomery, TX) (#A301-985A100, 1:100); Mouse Monoclonal anti-KAT3B/p300 (Abcam, Cambridge, UK) (#ab14984, 1:100); Normal mouse IgG (Santa Cruz, Santa Cruz, CA) (#sc-2025, 1:200); Normal rabbit IgG (Cell Signaling Tech, Danvers, MA) (#2729, 1:200) overnight. Antibody complexes were then captured with Dynabeads Protein G (Thermo Fisher, 10004D), and DNA was eluted, de-crosslinked, and purified. ChIP signal were calculated by qPCR (Supplementary Table 4) relative to input levels after (IgG) background subtraction.

Minichromosomes were isolated essentially as described^{46 (link)}. Cells carrying minichromosome plasmids were grown in 1 L of SILAC media^{70 (link)} (Kaiser SC-Arg-Lys-Trp (Formedium), supplemented with 20 mg/L heavy arginine-HCl (Arg10, CK Isotopes) and 30 mg/L lysine-2HCl (Lys6, CK Isotopes) or, for a control, 20 mg/L light arginine-HCl and 30 mg/L light lysine-2HCl) at 30 °C until log phase and harvested. After washing cells in ice-cold water supplemented with 2 mM PMSF, cells were resuspended in buffer H150 (25 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2 mM MgCl₂, 10% glycerol, 0.02% NP40, 1 mM PMSF, cOmplete Protease Inhibitor cocktail without EDTA (11873580001, Merck), PhosSTOP (4906845001, Merck), 5 mM nicotinamide) and disrupted using a FastPreP-24 bead beater (MP Biomedicals). Clarified lysates were prepared by centrifugation at 20,000 × g for 20 min three times. lacO-containing minichromosomes were isolated by immunoprecipitating LacI-3FLAG with anti-FLAG M2 antibody (12 μg, F1804, Merck) crosslinked to Dynabeads protein G (10765583, Fisher Scientific) by dimethyl pimelimidate. Proteins on purified minichromosomes were eluted in Elution buffer (50 mM ammonium bicarbonate, 0.1% RapiGest SF (186001860, Waters), 1 mM IPTG). The eluted proteins were analysed by SYPRO Ruby staining and western blot analysis.

After zFRAP analysis and observation, the zygotes were fixed with 4% paraformaldehyde containing 0.2% Triton X-100 for 20 min. After washing three times with PBS containing 1% BSA and 0.2% Tween 20, the zygotes were incubated with primary antibodies against H3K9me3 (ab8898; Abcam, Cambridge, MA, USA; 1:2000), Anti-H3.3 antibody (Clone 4H2D7, Cat# CE-040B, Cosmo Bio Co., LTD; 1:500), Anti-Flag M2 antibody (F1804, Merck, Darmstadt, Germany; 1:500), Anti-Hira (#39558, Active motif, Carlsbad, CA, USA; 1:500) diluted in PBS containing 1% BSA and 0.1% Triton X-100 at 4 °C overnight. After washing three times with PBS containing 1% BSA and 0.2% Tween 20, the zygotes were incubated with secondary antibodies (Alexa 568 conjugated anti-rabbit IgG, Alexa 488 conjugated mouse IgG; 1:500). The stained zygotes were mounted on PBS containing 4’,6-diamidino-2-phenylindole or Vectashield mounting medium with DAPI (Vector laboratories, INC.). The images were obtained by using confocal microscopy FV1200.

Monoclonal antibodies for Vasa, Siwi, and Ago3 detection were produced as described previously (Nishida et al, 2015). The anti‐Vret monoclonal antibody was raised in our laboratory (Nishida et al, 2020). We produced the anti‐DDX43 monoclonal antibody by immunizing mice with the purified GST‐tagged N‐terminal region (amino acids 1–200) of DDX43. The monoclonal ANTI‐FLAG M2 antibody (Merck, Darmstadt, Germany) for immunoprecipitation, anti‐DDDDK‐tag monoclonal antibody (FLA‐1 clone, Medical and Biological Laboratories, Aichi, Japan) for Western blotting and non‐immune mouse IgG (Immuno‐Biological Laboratories, Gunma, Japan) were commercially sourced.