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. 2013 Aug 2;3:2344. doi: 10.1038/srep02344

Figure 2. BRAF with constitutively active mutation is linked with Lys63-linked polyubiquitin chains.

Figure 2

(A) BRAF with constitutively active mutation was linked with HA-Ub-K63-only. The expression vector encoding full length FLAG-BRAF WT or constitutively active V600E or K601E mutant was co-transfected into HEK-293T cells with HA-Ub-K63-only expression vector. Lys 63-polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. (B) BRAF with V600E constitutive active mutation was modified by Lys63- but not Lys48-linked polyubiquitination. Empty vector or expression vector encoding HA-Ub-K63R or -K48R was co-transfected into HEK-293T cells with FLAG-BRAF. Ubiquitinated FLAG-BRAF proteins were detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. (C) BRAF with constitutively active mutation was endogenously modified by Lys63-linked polyubiquitination. The expression vector encoding full length FLAG-BRAF WT, constitutively active V600E or K601E mutant was transfected into HEK-293T cells. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-FLAG antibodies and immunoblotting with anti-Ub-K63-specific antibodies. (D) BRAF ED mutant was modified by Lys63-linked polyubiquitin chains compared with BRAF WT or BRAF AA mutant. Expression vector encoding FLAG-BRAF WT, AA and ED were co-transfected into HEK-293T cells with HA-Ub-K63-only vector, respectively. Lys63-linked polyubiquitinated FLAG-BRAF was detected by immunoprecipitation with anti-HA antibodies and immunoblotting with anti-FLAG antibodies. Cropped blots were used under the same experimental conditions.