GRA15-mediated parasite growth reduction in IFNγ-stimulated HFFs is independent of its ability to activate NF-κB but dependent on GRA15's interaction with the E3 ubiquitin ligases TRAF2 and TRAF6 A) HFFs were stimulated with IFNγ for 24 h (10 U/ml) or left unstimulated.Figure 4A) HFFs were stimulated with IFNγ for 24 h (10 U/ml) or left unstimulated. The NF-κB inhibitor BAY11-7082 (1µM) was added 2 h pre-infection and HFFs were subsequently infected with Pru parasites. Parasite growth (using luciferase assay) was measured 24 h p.i. Means from unstimulated cells were set at 100%. Experiments were performed three times. B) Nuclear translocation of the NF-κB p65 subunit was quantified in HFFs or HFFs treated with BAY11-7082 (1µM, added 2 h pre-infection) 24 h p.i. with Pru parasites. Experiments were done 4 times where each dot represents one experimental mean of at least 15 nuclei. In the right panel, representative images are shown. Parasites were expressing GFP, nuclei are stained with Hoechst 33258. Scale bar is 20 µm. C) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using an RH strain expressing type II GRA15-HA and as a control RH expressing GRA35-HA. The blots using antibodies against TRAF2 and TRAF6 were made after stripping the first blot. The inputs loaded represent 1% of total lysate prepared for immunoblotting and mass spec (Table EV1). The antibodies against TRAF2 and TRAF6 were obtained from Santa Cruz Biotechnology (Appendix Table S2). Full length blots for this figure can be observed in the source data for this figure. D) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using an RH strain expressing type II GRA15-HA and as a control RH expressing GRA43-HA. The blots using antibodies against TRAF6 were made after stripping the first blot that is used for HA blotting. The inputs loaded represent 10% of total lysate prepared for immunoblotting. Following TRAF6 blotting, the membrane was stripped and blotted for TNFAIP3. E) Immunofluorescence analysis of TRAF6 recruitment to the PVM of HFFs infected for 3 h with RH, Pru and PruΔgra15 strains. On the right-hand side, a representative fluorescent image is shown of TRAF6 recruitment to the PVM where Toxoplasma Pru strain expresses GFP, DNA was stained with Hoechst 33258. Scale bar is 10 µm. n=3 for all the strains. The antibody against TRAF6 was purchased from Abnova (Appendix Table S2). F) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using a RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut. For the unstimulated cells, uninfected HFFs were used an additional negative control (left panel). Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes. Full length blots are in the source data for this figure. The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova, respectively. The asterisks (*) in the lower right panel indicates the faint band of TRAF6 in the RH+GRA15TRAF2mut immunoprecipitate. Immunofluorescence analysis of p62, with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains. Immunofluorescence analysis of LC3B with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains. Immunofluorescence analysis of LAMP1 with and without IFNγ in RH, RH+GRA15WT. All the experiments were done 3 times with each of the strains. J) Plaque assays were performed with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3). K) Expression of TRAF6 was detected by Western blotting of lysates from scrambled siRNA transfected and TRAF6-specific siRNA transfected HFFs with and without IFNγ. L) Relative parasite growth was measured in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs using luciferase-based assay with indicated strains with and without IFNγ (n=3). The antibody against TRAF6 used here was from Abcam (Appendix Table S2). Full length blots are in supplementary figures 4l-m. Immunofluorescence analysis of p62 was done in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs with and without IFNγ using indicated strains (n=3). Immunofluorescence analysis of LC3B was done in scrambled siRNA transfected HFFs and TRAF6 knockdown HFFs with and without IFNγ using indicated strains (n=3).