Fig. 4: NLRP6 recruits RBX1 to ubiquitinate p85α.

a Coimmunoprecipitation (Co-IP) and immunoblot analysis of HEK293T cells transfected with Myc-NLRP6 and indicated plasmids. EV, empty vector, WCL, whole cell lysates. b Co-IP analysis of the interaction between NLRP6 and RBX1. c Co-IP analysis of the interaction between p85α and RBX1 in the presence of NLRP6 in HEK293T cells. d p85α-associated ubiquitins (UBs) were analysed in HEK293T cells transfected indicated plasmids with bafilomycin A1 (BafA1, 0.2 μM). e Endogenous p85α in LN229 cells transfected with indicated plasmids were analysed (top) and were quantified (bottom). f p85α-associated UBs were analysed in Control (Ctrl) or RBX1 knockout (KO) LN229 cells transfected with Myc-NLRP6. g Endogenous p85α in Ctrl, SKP1 KO, or RBX1 KO LN229 cells transfected with increasing amounts of Myc-NLRP6 were analysed. h Co-IP analysis of the interaction between p85α and RBX1 in Ctrl and NLRP6 KO LN229 cells. i Representative confocal microscopy of LN229 cells transfected with NLRP6 showing p85α and RBX1 colocalization (left). Scale bar = 10 μm. Pearson’s coefficient of colocalization was calculated (right). j p85α-associated UBs in Ctrl or NLRP6 KO LN229 cells transfected with Myc-RBX1 were analysed. k Endogenous p85α in Ctrl or NLRP6 KO LN229 cells transfected with increasing amounts of Flag-RBX1 were analysed. l Co-IP and immunoblot analysis of extracts from Ctrl or RBX1 KO LN229 cells transfected with indicated plasmids. m Co-IP and immunoblot analysis of LN229 cells transfected with indicated plasmids. In e, all error bars, mean values ± SD, p-values were determined by unpaired two-tailed Student’s t test of n = 3 independent biological experiments. For i, error bar, mean value ± SD, p-value were determined by unpaired two-tailed Student’s t test of n = 14 cells. Data are representative of three independent experiments with similar results (a–d, f, h, and j–m), or two independent experiments (g). Source data are provided as a Source Data file.