Ab23366

Western blot analysis was performed as previously described [7 (link)]. Antibodies specific to human proteins were anti-RUNX3 (ab40278, Abcam), anti-G9a (#3306, Cell Signaling Technology), and β-actin (SC-47778, Santa Cruz), pan-methyl lysine antibody (ab23366, Abcam).

Thin smears were prepared from Plasmodium falciparum culture at ring, trophozoite and schizont stages. The glass slides were air dried and fixed with pre-chilled absolute methanol for 30 minutes. The smears were incubated with 4% BSA in PBS for 2 h at room temperature (RT) to block the non-specific binding. After washing with PBS, the smears were probed with anti-mono/dimethyl lysine polyclonal antibody (abcam; ab23366)(1:20) for overnight at 4 °C, followed by Alexa-Fluor 488 conjugated goat anti-rabbit IgG antibody (A11008; Thermofisher Scientific Inc. USA) (1:200) for 1 h at RT. The slides were washed and mounted with 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI, Molecular Probes, USA) antifade solution (Molecular Probes, USA). Images were captured using a Nikon A1-R confocal microscope.

Cell extracts were prepared in RIPA buffer (50 nM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 0.25% deoxycholate) supplemented with protease inhibitor tablets (Roche Molecular Biochemicals) and phosphatase inhibitors (1 mM NaF, 1 mM Na₃VO₄, and 1 mM β-glycerophosphate). Protein extracts were incubated overnight at 4 °C with shaking with 1 μg of antibodies against HA epitope (Roche Applied Science 3F10) or pan methyllysine (AbCam ab-23366). Protein A/G plus agarose beads (Santa Cruz sc-2003) were added, and the mixture was incubated for 2 h at 4 °C. The immunoprecipitates were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Immunoblots were performed with primary antibodies against G9a (Sigma G6919), β-actin (Sigma A5441), GLP (Millipore 09-078), HP1γ (Abcam ab10480), cleaved Caspase 3 (Cell Signaling 9664S), cleaved Caspase 7 (Cell Signaling 8438S), cleaved PARP1 (Cell Signaling 9541S), HA epitope (3F10 Roche Applied Science), H3K9me2 (Cell signaling 4858S), histone H3 (Cell signaling 4499), GAPDH (Sigma G9545) or Flag (Sigma F1804). Secondary antibodies from Promega were used for chemiluminescence detection using ECL prime detection reagent (Amersham) according to the manufacturers’ instructions.