Anti flag m2 peroxidase

The following antibodies were used for Western blot analysis: rabbit β-tubulin (Cell Signaling Technology, 2146S), goat anti-rabbit IgG (H+L)-HRP Conjugate (Bio-Rad, 170-6515), ANTI-FLAG M2-Peroxidase (Sigma Aldrich, A8592), Apoptosis Western Blot Cocktail (abcam, ab136812), rat anti-LANA (Advanced Biotechnologies Inc., 13210), mouse anti-EBNA1 (Biorad), rabbit anti-HER2 (D8F12) (Cell Signaling), mouse monoclonal anti-actin-HRP (Sigma).

Cells were lysed in lysis buffer and the soluble fractions were collected and incubated with anti-HA angarose, anti-FLAG agarose (Sigma-Aldrich), or anti-symmetric dimethyl arginine antibody Sym10 (Upstate) conjugated with Dynabeads protein G magnetic beads (Invitrogen) for 2 h at 4°C. The precipitates were then washed three times with lysis buffer and boiled for 5 min in SDS loading buffer. Samples were analyzed by SDS-PAGE, transferred to Immobilon-P (Millipore) PVDF membrane, and probed with anti-Flag M2-Peroxidase (Sigma), or anti-HA Peroxidase (3F10; Roche). For the detection of tag proteins, immunocomplexes were detected using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore). For human Tregs, expanded cells were harvested and lysed on ice for 1 h with RIPA buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% NonidetP-40, 0.25% NaDOC, 1 mM MgCl₂, and 10% (vol/vol) glycerol) containing protease inhibitor (1:100; P8340; Sigma-Aldrich), NaF (10 mM), and PMSF (1 mM). Cell lysates were cleared by centrifugation, and the supernatants were incubated with anti-FOXP3 (1 μg, eBio7979), anti-PRMT5 (1 μg, Millipore O14744), or IgG (1 μg, 5415S; Cell Signaling) at 4°C overnight, and then immunoprecipated with Protein G-Sepharose beads (P3296; Sigma) for 1 h at 4°C. The immunocomplexes then were washed with RIPA buffer and examined by Western blotting.

Protein samples were denatured by boiling at 95°C for 3 min in protein-loading buffer [2% (w/v) SDS, 100 mM DTT, 0.05% (v/v) BPB, and 10% (v/v) glycerol], resolved by SDS-PAGE, and transferred to 0.2-μm polyvinylidene difluoride membrane (Wako) using the semi-dry system (Trans-blot Turbo, Bio-Rad). The membrane was blocked in 4% (w/v) skim milk (Nacalai) in 1× phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 and further incubated with the following antibodies: anti-Aub antibody (1:500; guinea pig) (80 (link)), anti-GFP antibody (1:2000; Clontech, rabbit), anti-Ago1 antibody (1:1000; Abcam, Ab5070, rabbit), anti-Ago2 antibody (1:100; guinea pig) (25 (link)), anti-Piwi (1:10; mouse, P4D2), anti-FLAG M2-peroxidase [horseradish peroxidase (HRP)] (1:5000; Sigma-Aldrich, #A8592), anti-H3K18Ac (1:2000; Active Motif, #39756), anti-H3K27Ac (1:2000; Active Motif, #39136), anti-H4K8Ac (1:2000; Active Motif, #61104), anti-H4K12Ac (1:2000; Active Motif, #39928), anti-guinea pig immunoglobulins-HRP (1:1000; Dako), anti-rabbit immunoglobulin G (IgG)–HRP (1:3000; Bio-Rad), anti-mouse IgG-HRP (1:3000; Bio-Rad). The chemiluminescent signals were obtained by using Chemi-Lumi One (Nacalai) and detected by Chemidoc MP Imaging system (Bio-Rad). The images were processed by using Pixelmator or Fiji. The immunoblot signals were quantified by using Fiji.