Extended Data Fig. 7: NLRP3 is recruited to dTGN via binding to PtdIns4P.

a, The polybasic region of NLRP3 interacts with phospholipids in vitro through its positive charge. Left, Coomassie blue staining of purified proteins of interest, with Flag–GFP as a control. Right, PIP Strip membranes blotted with various lipids were incubated sequentially with proteins of interest, Flag antibody and HRP secondary antibody before exposure. Phospholipids with positive binding on the second PIP Strip are highlighted in red. b, Catalytically inactiveSac1 did not impair the recruitment of NLRP3 to dTGN. COS-7 cells stably expressing Flag-NLRP3 were transiently transfected with TGN38–FRB and mRFP–FKBP12–Sac1(C389S), incubated with rapamycin (1 μM) and nigericin (10 μM) for 80 min before imaging. Note that nigericin-induced dTGN in COS-7 cells sometimes looks like a single cluster because of the relatively small size of COS-7 cells and the high intensity of fluorescence signal. Under the phase contrast microscope these dTGN particles were observed to be individual vesicles adjacent to each other (data not shown). c, Only PtdIns4P phosphatase blocked NLRP3 recruitment to dTGN. Similar to b, except that indicated phosphatases were used. The target phospholipids are labelled after ‘x’. d, Immunoblots of HeLa NLRP3–GFP cells stably expressing TGN38–Sac1 or TGN38–Sac1(C389S). e, TGN-targeted Sac1 did not affect general cell morphology or nigericin-induced dTGN formation. Cells were treated with nigericin (10 μM) for 80 min before imaging with phase contrast microscopy. Cells with dTGN formation were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). n.s., not significant (α = 0.01). f, TGN-targeted Sac1 did not affect AIM2 aggregation. Cells stably expressing the indicated proteins were transfected with poly(dA:dT) (1.5 μg ml⁻¹) for 3 h before imaging. The percentage of cells with AIM2 aggregates was analysed as in e. g, NLRP3 puncta were restricted to PtdIns4P-enriched microdomains. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 80 min before imaging. Inset, higher magnification of the dTGN. Co-localization analysis was performed by calculating Pearson's correlation coefficient (threshold regression, Costes) using the Coloc 2 plugin of ImageJ. Data are presented as mean ± s.d.