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. Author manuscript; available in PMC: 2021 Nov 8.
Published in final edited form as: Circ Res. 2020 Mar 16;126(10):1379–1393. doi: 10.1161/CIRCRESAHA.119.315730

Figure 4. LZTR1 regulates CHMP1B ubiquitination.

Figure 4.

(a) Ubiquitinome analysis of wt-LZTR1 or LZTR1-indel HeLa cells. N=3. For each protein, log2 (fold change between both conditions) is plotted vs Fold Change Rank. CHMP1B ubiquitination sites are shown in red. (b, c) An empty vector (EV), HA-tagged wt-LZTR1, or LZTR1 mutants were overexpressed in HeLa cells. Endogenous CHMP1B was immunoprecipitated using anti-CHMP1B antibody conjugated to agarose. The amount of pulled-down LZTR1 was detected by immunoblotting with anti-HA antibody. (d) CHMP1B was pulled down from ECs expressing HA-wt-LZTR1 using an anti-CHMP1B antibody. The amount of pulled-down HA-wt-LZTR1 was detected by immunoblotting. (e) Ubiquitinated proteins were immunoprecipitated from Lztr1+/+ and Lztr1+/− ECs isolated using anti-ubiquitin FK2 antibody. The amount of pulled-down CHMP1B was detected by immunoblotting. (f) Ubiquitinated proteins were immunoprecipitated from ECs treated with VEGF (100ng/ml, 30 minutes) or MLN4924 (1μM, 12hours) using anti-Ubiquitin FK2 antibody. The amount of pulled-down CHMP1B was detected by immunoblotting. (g) Ubiquitinated proteins were immunoprecipitated from HeLa cells with Lztr1-indel overexpressing empty vector (EV), wt-LZTR1, or NS-associated LZTR1 mutants using anti-ubiquitin FK2 antibody conjugated to agarose. The amount of pulled-down CHMP1B was detected by immunoblotting. * indicates ubiquitinated CHMP1B.