Ab9106

Cells in supplemented EMM were collected and whole-cell protein extract was prepared by vortexing acid-washed glass beads in 20% trichloroacetic acid (TCA) and washing beads with 5% TCA. Lysates were boiled for 5 min in Laemmli Sample buffer (4% SDS, 60 mM Tris-HCl, pH 6.8, 5% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and analyzed by SDS-PAGE. Primary antibodies used were as follows: anti-phospho-H2A (Abcam ab17353; 1:1,000), anti-H2A (Cell Signaling 3636S; 1:1,000; re-probed after phospho-H2A), anti-GFP (Abcam ab291; 1:1,000), anti-myc (Abcam ab9106; 1:1,000) anti-PCNA (Santa Cruz sc-56; 1:1,000), and anti-cdc2 (Abcam ab5467; 1:1,000). After secondary antibody (Alexa Flour 488 or 647; 1:6,000) incubation, blots were developed using Amersham Typhoon biomolecular imager. Intensity of bands were quantified with ImageJ.

Intracellular tachyzoites grown in cover slips were fixed using cold methanol 100% for 8 min, washed with PBS and blocked with 1% BSA. Primary antibody αrH2B.Z (rabbit), αrSag1 (mouse), αrHSP90 (rabbit), αrROP5 (rabbit), αrROP18 (rabbit) produced in the laboratory and αc-Myc (rabbit, abCam ab9106 or mouse, Santa Cruz sc-42), αAcTubulin (mouse, Millipore MABT868) were incubated for 1 h at room temperature. After several washes with PBS cover slips were incubated with secondary antibodies Alexa fluor goat anti-mouse 488 and anti-rabbit 594 (Invitrogen). DAPI was used to stain nuclei. Axio Imager.M2 Microscope Carl Zeiss (Germany) with objective Plan-Apochromat 63x/1.40 Oil M27 and Video digital camera Zeiss 503 monochromatic 2.8 megapixeles, was used. Image J 1.53q (Fiji) software was used to process images.