Phosphatase conjugated goat secondary antibodies

For histone purification, HFF cells were grown to confluence and infected with Pru∆ku80 parasites. Intracellular tachyzoites were treated with histone deacetylase HDAC3 inhibitor, 90 nM FR235222 for 18 hr. As an appropriate control, we treated tachyzoites with 0.1% DMSO. Histones were extracted and purified using histone purification kit (Active motif) according to manufacturer’s protocol. For western blotting, histone proteins were run on a NuPAGE 4–12% Bis-Tris polyacrylamide gels in MES-SDS running buffer (Invitrogen) and transferred to a polyvinylidene fluoride PVDF membrane (Immobilon-P; Millipore) using NuPAGE transfer buffer (Invitrogen). The blots were probed using primary antibodies: pan acetyl H4, H4K31ac and H4K31me1, followed by phosphatase-conjugated goat secondary antibodies (Promega). The expected band of histones were detected using NBT-BCIP (Amresco). Nucleosomes from T. gondii-infected cells were purified and proteins separated by SDS-PAGE. The band corresponding to H4 was excised and its protein content digested using trypsin. The resulting peptides were submitted to mass spectrometry-based proteomic analysis (U3000 RSLCnano coupled to Q-Exactive HF, Thermo Scientific). Peptides and proteins were identified using Mascot software (Matrix Science).