Fig 2: Rictor inhibited NLRP3 transcription through increasing the K48‐linked ubiquitination of p65 by E3 ubiquitin ligase SOCS1. a) Western blotting analysis of total and phosphorylated p65 in WT or Rictor‐/‐BMDMs treated with LPS (200 ng mL−1) for indicated time. b) Western blotting analysis of indicated proteins in WT or Rictor‐/‐BMDMs priming with LPS (200 ng mL−1) and JSH‐23 (20 µm) for 4 h. c) qRT‐PCR analysis showing the mRNA level of p65 in WT or Rictor‐/‐ BMDMs treated with LPS (200 ng mL−1) for 4 h (n = 5). d) Luciferase activity in negative control (NC) or Rictor overexpression (OE) HEK293T cells transfected with plasmids encoding an NF‐κB luciferase reporter and TK‐Renilla reporter, together with indicated plasmids, was measured at 24 h after transfection and normalized to the Renilla luciferase activity (n = 3). e) Western blotting analysis of p65 in NC and Rictor OE HEK293T cells were treated with CHX (200 µg mL−1) for indicated time. f) Decay curve of p65 normalized to β‐actin and to 0 h at indicated time points from CHX‐chase assay (n = 3). g) Western blotting analysis of p65 in NC or Rictor OE HEK293T cells transfected with Flag‐p65 and treated with MG132 (10 µm) for 6 h or CQ (10 µm) for 24 h. h) Co‐immunoprecipitation (IP) assay for the ubiquitination of p65 in NC or Rictor OE HEK293T cells transfected with HA‐p65 and treated by MG132. i) Co‐IP assay for the ubiquitination of p65 in NC or Rictor OE HEK293T cells transfected with the indicated plasmids and treated by MG132. j–n) qRT‐PCR analysis showing the mRNA levels of PDLIM2 (j), Trim21 (k), RNF182 (l), SOCS1 (m) and Trim7 (n) in WT or Rictor‐/‐ BMDMs treated with LPS (200 ng mL−1) for 4 h (n = 5). o) HEK293T cells transfected with plasmids encoding an NF‐κB luciferase reporter and TK‐Renilla reporter, together with Flag‐p65 and indicated siRNAs, were measured at 24 h after transfection and normalized to the Renilla luciferase activity (n = 3). p) Co‐IP assay for the ubiquitination of p65 in NC or Rictor OE HEK293T cells transfected with the HA‐K48 and si‐SOCS1 and treated by MG132. q) A schematic model for this study. Data were analyzed using unpaired Student's t‐test. * p < 0.05; ** p < 0.01. Data are presented as mean ± SD.