Ab177253

Cultured T. annulata-infected macrophages (TaC12) were washed with PBS containing 1 mM EDTA and 3 × 10⁴ cells per slide were centrifuged with Cytospin (10 min at 277 g) to adhere to the slide. Cells were fixed in 3.7% paraformaldehyde for 15 min and subsequently permeabilized in 0.2% Triton X-100 (prepared in PBS) for 10 min. Fixation, permeabilization and all the following steps were carried out at room-temperature. Slides were blocked with PBS 0.2% Tween (PBST)–1% BSA for 30 min. Rabbit anti-H3K18me1 (ab177253, Abcam, 1:5000 dilution) antibodies were diluted in PBST and incubated for 1 h. Cells were subsequently washed three times with PBST and incubated with secondary antibody for 30 min with Alexa594-conjugated donkey anti-rabbit antibody (1:500 dilution). Cells were washed three times with PBST and finally, mounted on coverslips adding ProLong Diamond Antifade Mountant implemented with DAPI counterstain (Thermo Fischer Scientific). Samples were analysed using a Leica DMI6000 epifluorescence microscope. Images were generated and processed using Metamorph and ImageJ software. Parasite load was quantified by counting parasite nuclei in the host cytoplasm by DAPI. The macroschizont or merogony stages were quantified using ImageJ. We defined a threshold for the Schizont/Merogony cycle stage of 50 parasites per host cell.

2 µg of 5-FAM coupled short peptides (Proteogenix) flanking the unmodified, monomethylated, dimethylated, trimethylated or acetylated lysine of interest (H3K4: ARTKQTARRSK, H3K9: RQTARKSTGG, H3K14: STGGKAPRR, H3K18: RAPRKQLAT, H3K27: TKAARKSAPAT and H3K36:
TGGVKRPHR) were transfered on nitrocellulose membrane using Bio-Dot^®microfiltration apparatus (Bio-Rad) and blocked with 5% non-fat milk in PBS-Tween for 1 h. Membranes were then incubated with antibodies against H3K18me1 (Abcam #ab177253, Active Motif #31259 and a home-made antibody provided by Jane Mellor’s laboratory) (1:10,000) or H3K36me3 (Abcam #ab9050) (1:10,000) at 4 °C overnight. Membranes were washed three times with PBS-Tween for 10 min and incubated with a 1:20,000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody for 1 h at room temperature. Membranes were finally developed with the Pierce ECL Western Blotting Substrate according to the manufacturer’s protocol.