HUWE1 regulates ubiquitination of Shoc2 and RAF-1.

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HUWE1 regulates ubiquitination of Shoc2 and RAF-1. HUWE1 regulates ubiquitination of Shoc2 and RAF-1. (A) Shoc2 was precipitated from HeLa cells using Shoc2 antibodies. Shoc2 ubiquitination was detected with anti-UB antibodies. Immunoprecipitates and lysates were analyzed by immunoblotting using anti-HUWE1 and anti-Shoc2 antibodies. (B) GST-Shoc2 was precipitated from HeLa cells transfected with HA-Ub using glutathione-Sepharose. Shoc2 ubiquitination was detected with anti-HA antibodies. Immunoprecipitates and lysates were analyzed by immunoblotting with anti-HUWE1 and anti-GST antibodies. (C) HeLa cells were transfected with YFP–RAF-1 and HA-Ub. Cells were treated with the proteasome inhibitor MG132 (10 μM). YFP–RAF-1 was precipitated using anti-green fluorescent protein (anti-GFP) antibodies that cross-react with YFP. RAF-1 ubiquitination was detected with anti-HA antibodies. Immunoprecipitates and lysates were analyzed by immunoblotting with anti-HUWE1 and anti-RAF-1 antibodies. (D) 293FT Shoc2-GST-expressing cells were cotransfected with WT HA-HECT or HA-HECT with the C4341A (C/A) substitution. GST-Shoc2 was precipitated using glutathione-Sepharose, and Shoc2 ubiquitination was detected with anti-Ub antibodies. (E) 293FT Shoc2-GST-expressing cells were transfected with the full-length HA-HUWE1. GST-Shoc2 was precipitated using glutathione-Sepharose, and Shoc2 ubiquitination was detected with anti-Ub antibodies. (F) 293FT cells were transfected with WT HA-HECT, HA-HECT with the C4341A substitution, or empty vector. RAF-1 was precipitated using anti-RAF-1 antibody, and RAF-1 ubiquitination was detected with anti-Ub antibodies. (G) 293FT cells were transfected with the full-length HA-HUWE1. RAF-1 was precipitated using anti-RAF-1 antibody, and RAF-1 ubiquitination was detected with anti-Ub antibodies. The results in each panel are representative of those from three independent experiments. Lanes MW, molecular weight markers (numbers to the left of the gels are in thousands). Eun Ryoung Jang et al. Mol. Cell. Biol. 2014;34:3579-3593