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. 2012 Dec 31;288(6):3753–3767. doi: 10.1074/jbc.M112.415240

FIGURE 6.

FIGURE 6.

A, overexpression of HA-HectD1 increased the pool of endogenous APC modified with Lys-63 polyubiquitin. HEK293 cells were transfected with Trabid siRNA (72 h) and expression vectors for WT or C2579G HA-HectD1 (48 h) as indicated. The cells were treated with vehicle (dimethyl sulfoxide, −) or 10 μm MG132 for 4 h. Polyubiquitin adducts in lysates were immunoprecipitated with Lys-63 polyubiquitin linkage-specific antibodies (control IgG immunoprecipitation, anti-E25). Immunoprecipitates and lysates were analyzed by immunoblot (IB) as indicated. B, RNAi-mediated depletion of HectD1 decreased the pool of endogenous APC modified with Lys-63 polyubiquitin. HEK293 cells were transfected (72 h) with siRNAs targeting individually or combinations of Trabid, HectD1, or all three Striatins as indicated, followed by treatment with vehicle (−) or 10 μm MG132 for 4 h. Lysates were immunoprecipitated as described in A and analyzed by immunoblot with the indicated antibodies. C, depletion of HectD1 decreased the APC-Axin interaction. HEK293 cells were transfected with the indicated siRNAs for 72 h. Lysates were immunoprecipitated using anti-APC or control mouse IgG antibody and analyzed by immunoblot as indicated.