VANGL2 recruits MARCH8 to promote the K27-linked ubiquitination of NLRP3.

(A) PEMs were silenced with <i>Vangl2</i> siRNA for 24 h, followed by LPS (100 ng/ml) and CQ (50 μM) treatment for 6 h, and NLRP3 was pulled down by IP. Immunoblot analysis was used to detect the expression of Ub and NLRP3. (B) HKE293T cells were silenced with <i>VANGL2</i> siRNA for 24 h, then transfected with HA-Ub-K27 and Flag-NLRP3 plasmids for 24 h, followed by CQ (50 μM) treatment for 6 h. Flag was pulled down by IP, and the expression of HA and Flag tagged proteins were detected by immunoblot analysis. (C–E) HKE293T cells were silenced with <i>MARCH1</i> siRNA (C), <i>MARCH3</i> siRNA (D), or <i>MARCH11</i> siRNA (E) for 24 h, and then transfected with Flag-NLRP3 and Myc-VANGL2 plasmids for 24 h. Subsequently, the expression of Flag and Myc-tagged proteins were detected by immunoblot analysis. (F) HEK293T cells were silenced with <i>MARCH7</i> siRNA for 24 h, followed by transfection with HA-Ub-K27, Flag-NLRP3, and Myc-VANGL2 plasmids for 24 h. Flag was then pulled down through IP, and the expression of HA, Flag, and Myc tagged proteins were detected by immunoblot analysis. (G) HEK293T cells were silenced with <i>MARCH7</i> siRNA for 24 h, followed by transfection with Flag-NLRP3 and Myc-VANGL2 plasmids for 24 h. The expression of Flag, Myc, and MARCH7 proteins were detected by immunoblot analysis. (H) PEMs were silenced with <i>March8</i> siRNA for 24 h, followed by LPS (100 ng/ml) treatment for 6 h, and finally the expression of NLRP3 was detected by immunoblot analysis. (I) PEMs were silenced with <i>March8</i> siRNA for 24 h, followed by LPS (100 ng/ml) treatment for 6 h and ATP treatment for 30 min. The expression of IL-1β was detected by ELISA. Data are expressed as means ± SD. ***<i>P</i> < 0.001. (PDF)