Fig. 3: Ubiquitination of p85α on K256 is critical for autophagic degradation.

a Coimmunoprecipitation (Co-IP) and immunoblot analysis of extracts of LN229 cells transfected with HA-p85α together with Flag-tagged wild type (WT) OPTN or ΔUBA-OPTN. WCL, whole cell lysates. b Co-IP and immunoblot analysis of extracts from Control (Ctrl) or NLRP6 knockout (KO) LN229 cells. c Co-IP and immunoblot analysis of extracts of HEK293T transfected with HA-UB and empty vector (EV), Flag-p85α (WT), Flag-p85α (ΔRHO), or Flag-p85α (RHO) in the presence of Myc-NLRP6. d Co-IP and immunoblot analysis of extracts of LN229 cells transfected with Myc-NLRP6 and empty vector (EV), HA-p85α (WT), HA-p85α (K224R), HA-p85α (K225R), HA-p85α (K245R), or HA-p85α (K256R) in the presence of BafA1 (0.2 μM). e Co-IP and immunoblot analysis of purified His-NLRP6 with GST-p85α (WT) or GST-p85α (K245R). f Mass spectrometry analysis of a peptide derived from p85α identifying the attachment of ubiquitin to K256. g Mass spectrometry analysis of a peptide derived from p85α identifying that ubiquitin was not attached to K245. h Immunoblot analysis of extracts of LN229 cells transfected with Flag-p85α (WT), Flag-p85α (K245R), and Flag-p85α (K256R) in the presence of Myc-NLRP6 (left). Quantification of the protein levels of p85α (right). i Co-IP analysis of the interaction between various p85α mutants and OPTN in HEK293T cells. In h, all error bars, mean values ± SD, p-values were determined by unpaired two-tailed Student’s t test of n = 3 independent biological experiments. Data are representative of three independent experiments with similar results (a, b, d, e, and i), or two independent experiments (c). Source data are provided as a Source Data file.