Mouse anti t gondii

T25 flasks of confluent HFFs were infected with 1 million parasites for 2 h, after which the HFFs were dissociated and fixed with methanol for 10 minutes. The cells were stained with 1:200 rabbit anti-phospho-STAT6 (Cell Signaling #56554) and 1:200 mouse anti-T. gondii (Santa Cruz #SC-52255) overnight at 4°C, followed by 1:1000 goat anti-rabbit 488 (Invitrogen #A11008), 1:1000 goat anti-mouse 594 (Invitrogen #A11005), and 5 μg/mL DAPI for 1 h at room temperature. Data were collected on an LSR II flow cytometer (BD) running FACSDiva v9 (BD) and analysed with FlowJo v10 (www.flowjo.com). The median anti-phospho-STAT6 signal in the infected cells was determined for each sample, and the median technical replicate taken to represent the biological replicate. The data were scaled to RHΔKU80 = 1 AU and differences between strains tested by two-sided Wilcoxon rank sum test with Benjamini-Hochberg adjustment.

HFFs were grown to confluence in an 8-well μ-slide (Ibidi) and serum starved for 24 h before infection in 0.1% FBS medium. Each well was infected with 40,000 parasites for 24 h in 0.1% FBS medium before fixation with 4% w/v formaldehyde for 15 min, permeabilisation with 0.2% v/v Triton X-100 for 15 min, and blocking with 2% w/v BSA for 1 h. The cells were stained with 1:800 rabbit anti-cMyc (Cell Signaling #5605) and 1:200 mouse anti-T. gondii (Santa Cruz #SC-52255) for 2 h at room temperature, followed by 1:1000 goat anti-rabbit 488 (Invitrogen #A11008), 1:1000 goat anti-mouse 594 (Invitrogen #A11005), and 5 μg/mL DAPI for 1 h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon Plan APO 40x/0.95 objective and Hamamatsu C11440 ORCA Flash 4.0 camera running NIS Elements (Nikon) and analysed using ImageJ [63 (link)]. The median cMyc fluorescence intensity in each nucleus was determined and the median nucleus taken as representative of a replicate. The median background cMyc fluorescence intensity was subtracted, and the data normalised to RHΔKU80 = 1 AU for each biological replicate. Differences between strains were tested by two-sided Wilcoxon rank sum test with Benjamini-Hochberg adjustment.

Parasites were purified from host cell material by syringe-lysis, filtering and washing in PBS, then lysed in RIPA buffer (Pierce) supplemented with 2x cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche). 10 μg protein per sample was boiled for 5 min in sample loading buffer and separated by SDS-PAGE using the Mini-PROTEAN electrophoresis system (Bio-Rad). Proteins were transferred to a nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad), blocked in 2% w/v skim milk powder, 0.1% v/v Tween 20 in PBS for 1 h at room temperature, then incubated with primary antibodies in blocking buffer overnight at 4°C. Primary antibodies used were 1:1000 mouse anti-ROP1 (Abnova #MAB17504), 1:1000 rat anti-HA (Roche #11867423001), and 1:200 mouse anti-T. gondii (Santa Cruz #SC-52255). Blots were stained with secondary antibodies for 1 h at room temperature: 1:10,000 goat anti-rat IRDye 680LT (Li-Cor #925–68029) and 1:10,000 goat anti-mouse IRDye 800CW (Li-Cor #925–32210). Blots were visualised using an Odyssey CLx scanner (Li-Cor).