VANGL2 recruits NLRP3 to autophagy receptor OPTN for selective autophagic degradation.

(A) Flag-ASC and HA-VANGL2 plasmids were transfected into HEK293T cells for 24 h, and the expression of Flag and HA-tagged proteins were detected by immunoblot analysis. (B) Flag-Casp1 and HA-VANGL2 plasmids were transfected into HEK293T cells for 24 h, and the expression of Flag and HA-tagged proteins were detected by immunoblot analysis. (C) LPS-primed PEMs (WT and <i>Vangl2</i>^ΔM/ΔM) were cultured in EBSS for 0–3 h. Immunoblot analysis was used to detect the expression of NLRP3 and VANGL2. (D) Flag-NLRP3 and HA-VANGL2 plasmids were transfected into WT and <i>p62</i> KO HEK293T cells, and the expression of Flag-NLRP3, HA-VANGL2, and p62 were detected by immunoblot analysis. (E) Flag-NLRP3 and HA-VANGL2 plasmids were transfected into WT and <i>TOLLIP</i> KO HEK293T cells, and the expression of Flag-NLRP3, HA-VANGL2, and TOLLIP were detected by immunoblot analysis. (F) Flag-NLRP3 and HA-VANGL2 plasmids were transfected into WT and <i>NDP52</i> KO HEK293T cells, and the expression of Flag-NLRP3, HA-VANGL2, and NDP52 were detected by immunoblot analysis. (G) Flag-NLRP3 and HA-VANGL2 plasmids were transfected into WT and <i>NBR1</i> KO HEK293T cells, and the expression of Flag-NLRP3, HA-VANGL2, and NBR1 were detected by immunoblot analysis. (H) HEK293T cells were silenced with scramble siRNA and <i>VANGL2</i> siRNA for 24 h, and then transferred with HA-NLRP3 and Flag-OPTN plasmids for 24 h, followed by treatment with CQ (50 μM) for 6 h. The expression of HA and Flag tagged proteins were detected by Co-IP and immunoblot analysis. (I) WT and <i>Vangl2</i>^ΔM/ΔM PEMs were pretreated with LPS (100 ng/ml) and CQ (50 μM) for 6 h, and then NLRP3 was pulled down by IP. The expression of LC3B and NLRP3 were detected by immunoblot analysis. (J) Flag-NLRP3, GFP-LC3B, and HA-VANGL2 plasmids were transfected into WT and <i>OPTN</i> KO HEK293T cells for 24 h, and then treated with CQ (50 μM) for 6 h. The expression of GFP, Flag, and HA tagged proteins were detected by Co-IP and immunoblot analysis. (PDF)