Fig. 1

(A) Proteasome and p97/VCP inhibitors inhibited the degradation of endogenous Vangl proteins. HEK293T cells were treated with CHX (100 μg/ml) alone or with proteasome inhibitor MG132 (10 μM), lysosome inhibitor CQ (25 μM) or p97/VCP inhibitor DBeQ (10 μM), NMS-873 (2 μM), or CB-5083 (5 μM) for the indicated time. Endogenous Vangl1 and Vangl2 were detected, and the relative Vangl protein levels were quantified below. (B) Knockdown of VCP by small interfering RNA (siRNA) increased Vangl protein levels in HEK293T cells. (C) Single guide RNA (sgRNA)–mediated knockdown of ERAD component HRD1 or MARCH6, but not SEL1L or DERL1, increased Vangl protein levels in HEK293T cells. The Vangl protein levels were quantified in the right panel. (D) Vangl2 was poly-ubiquitinated. Treatment of proteasome inhibitor MG132 (10 μM) for 4 hours significantly increased Vangl2 ubiquitination. (E) Vangl2 underwent K48-linked poly-ubiquitination. HEK293T cells were treated with MG132 (10 μM) or CQ (25 μM) for 4 hours, and ubiquitination was examined by total ubiquitin FK2, K48 linkage–, or K63 linkage–specific antibodies. The bands at 55 and 100 kDa were immunoglobulin G and nonspecific bands, respectively. Ub, ubiquitin. (F) K300 and K306 contribute to Vangl2 ubiquitination. The corresponding Vangl2 lysines (K) were mutated to arginines (R), which reduced the level of Vangl2 ubiquitination. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.