Mab17504

HFFs were grown to confluence in an 8-well μ-slide (Ibidi) and infected with T. gondii strains for 24 h. The slides were fixed with 4% w/v formaldehyde (Sigma) in phosphate-buffered saline (PBS) (Sigma). The cells were permeabilised with 0.2% v/v Triton X-100 (Sigma) for 15 min or 1 min and blocked with 2% w/v bovine serum albumin (Sigma) for 1 h. The cells were stained with 1:500 rat anti-HA (Roche #11867423001), followed by 1:1000 goat anti-rat 594 (Invitrogen #A11007), followed by a mixture of 1:500 mouse anti-ROP1 (Abnova #MAB17504) and 1:1000 rabbit anti-T. gondii (Abcam #ab138698), and finally with a mixture of 1:1000 goat anti-mouse 488 (Invitrogen #A11029), 1:1000 goat anti-rabbit 647 (Invitrogen #A21244), and 5 μg/mL DAPI (Sigma), each for 1h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon CFI APO TIRF 100x/1.49 objective and Hamamatsu C11440 ORCA Flash 4.0 camera running NIS Elements (Nikon).

For Western blotting analysis, the samples were lysed in 300 μL IP buffer and incubated with 30 μL Pierce anti-HA magnetic beads (Thermo) overnight at 4°C, following which the beads were washed three times with IP buffer and the bound proteins eluted with 30 μL 3x Sample Loading Buffer (NEB) at 95°C for 10 min. 5% of the post-IP lysate supernatant and 15% of the immunoprecipitate were separated by SDS-PAGE and transferred to a nitrocellulose membrane as above. The membrane was blocked with 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 rat anti-HA (Roche #11867423001), 1:1000 mouse anti-ROP1 (Abnova #MAB17504), 1:1000 rabbit anti-C1QBP (Abcam #ab270032), 1:10,000 rabbit anti-GAPDH (Proteintech #10494-1-AP), and 1:1000 rabbit anti-GRA29 [15 (link)]. Blots were stained with secondary antibodies for 1 h at room temperature: 1:10,000 goat anti-mouse IRDye 680LT (Li-Cor #925–68020), 1:10,000 goat anti-rat IRDye 800CW (Li-Cor #925–32219), 1:10,000 donkey anti-rabbit IRDye 680LT (Li-Cor #925–68023), and 1:10,000 donkey anti-rabbit IRDye 800CW (Li-Cor #925–32213). Blots were visualised using an Odyssey CLx scanner (Li-Cor).