Ab18255

The following antibodies were used in this study: rabbit polyclonal antibodies against PCNA (ab15497, Abcam), histone H2A (ab18255, Abcam) and StrepII (ab76949, Abcam); mouse monoclonal antibodies against FLAG (Sigma, M2) and ubiquitin (ab7254, Abcam); and DyLight^™ 800-conjugated Streptavidin (S000-32, Rockland).

Chromatin samples were fractionated on a 4–14% SDS-PAGE gel, transferred to nitrocellulose membrane using Towbin buffer (25 mM Tris pH 8.8, 192 mM Glycine, 20% methanol) on a Hoefer TE 77 semi-dry transfer unit for 2 h at 55 mA/membrane. The membrane was incubated in blocking solution (5% non-fat milk in TBS) for 1 h prior to replacement with primary antibody diluted in TBS with 1.5% non-fat milk: anti-Histone H3 (abcam ab1791, 1:10,000), anti-Histone H2B (abcam ab1790, 1:1000), anti-Histone H2A (abcam ab18255, 1:1000), anti-Histone H4 (abcam ab10158, 1:1000), anti-Histone H3K18ac (Active Motif 39,755, 0.5 μg/ml) or anti-Histone H3K27me2 (Active Motif 39,245, 1:1000). Membrane was incubated on a rocker overnight at 4 °C, washed in TBS-T (TBS, 0.1% Tween-20) three times for 10 min and incubated in TBS with 1.5% non-fat milk containing a secondary goat anti-rabbit IgG Alexa Fluor conjugated antibody (Life Technologies A27042, 1:15,000) for 45 min at room temperature. The membrane was washed in TBS-T three times for 10 min, rinsed in diH₂O and visualized using a LI-COR Odyssey. Membrane was then stained with 0.1% amido black 10B (Sigma N3393) in 10% acetic acid for 1 min, destained with 5% acetic acid twice for 1 min and rinsed in diH₂0 twice for 10 min before imaging using a LI-COR Odyssey.

Part of the tissues of TT and TP in PA patients was homogenized in lysis buffer containing protease and phosphatase inhibitor (Sigma-Aldrich). Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose membrane. After blocking, incubate with TRPM2, GAPDH, Ras, Raf-1, PSPH, OASL, PKC, METTL3, HIST1H2AE, cPLA, and AQR antibodies (abcam, ab11168, ab9482, ab52939, ab173539, ab211418, ab229136, ab205791, ab195352, ab18255, ab53421, ab205303) at 4 °C overnight. After incubating with the secondary antibody at 20 °C for 1 h, the membrane was washed 3 times. The immune response zone is detected by the ECL detection system. The protein bands were quantified using Image J software (NIH, Bethesda, MD, USA).

Rabbit polyclonal anti-H2A (Abcam ab18255) and donkey-anti rabbit (711-005-152; Jackson ImmunoResearch) secondary antibodies were labeled in-house with different combinations of pairs of activator/reporter dyes (14 (link)). Briefly, dyes were purchased as NHS ester derivatives: Alexa Fluor 405 Carboxylic Acid Succinimidyl Ester and Alexa Fluor 647 Carboxylic Acid Succinimidyl Ester (Invitrogen). Labeling reactions were performed by incubating at room temperature for 40 min a mixture containing the secondary antibody in 0.12 M NaHCO₃, and the appropriate pair of activator/reporter dyes diluted in DMSO. Purification of labeled antibodies was performed using NAP5 Columns (GE HealthCare). The dye to antibody ratio was quantified using Nanodrop and only antibodies with a composition of 3–4 Alexa Fluor 405 and 0.9-1.2 Alexa Fluor 647 per antibody molecule were used for imaging.
Extracellular vesicles in H2B-GFP BMDMs were analyzed by STORM with the same protocol without the permeabilization step to avoid excessive staining of the nucleus. The cell membranes were stained with 50 µg/ml Concanavalin A-TRITC (Molecular Probes, Eugene, OR, USA).

Whole worm lysates were generated from indicated worms. ~100 worms were collected, washed in M9 buffer and resuspended in equal volume of 2X Laemmli sample buffer (Bio-RAD). Worm lysates or E3 ligase reaction mixtures were resolved on 4–20% stain-free SDS-PAGE gels (Bio-RAD) and transferred to Millipore Immobilon-P PVDF membranes. Membranes were blocked with 5% nonfat milk and probed with mouse anti-FLAG (MA1-91878; Invitrogen; 1:1000; RRID AB_1957945), rabbit anti-GFP (NB600-308; Novus Biologicals; 1:2000; RRID: AB_10003058), mouse anti-HA [12CA5; amino acids 98–106 of human influenza virus hemagglutinin protein; IgG2b mAb; 1:1000; RRID: AB_2532070; in-house (Trimmer Laboratory)], or anti-Histone-H2A (ab18255; Abcam; 1:1000; RRID:AB_470265) followed by IRDye800-conjugated anti-mouse IgG secondary antibodies (962 32212; LI-COR Bioscience; 1:20000; RRID: AB_621847). Immunoblots were imaged on a LI-COR Odyssey Infrared Imager, signal was quantified using Fiji and normalized with total protein stain.