Ube2d1

Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 20X E1 Enzyme, 10X Mg2+-ATP Solution, 10X Ubiquitin Solution, 1ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-YAP antibody (#14074, Cell signaling, 1:1000).

YAP and FBXW7 were cloned into the pET26b vector. OTUB1 were cloned into the pGEX-4T1 vector. Protein purification followed the general procedure in the pET System Manual. Ubiquitination was performed with the ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were incubated with 20X E1 Enzyme, 10X Mg²⁺-ATP Solution, 10X Ubiquitin Solution, 1 ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer at 37 °C for 1 h. Finally, products were analyzed by western-blot assays.

Ubiquitination was analyzed with a ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. The recombinant proteins MDM2 and P53 were acquired from Sangon Biotech (China). The RNF187 protein was expressed in and purified from HEK293 cells with anti-Myc beads. Recombinant proteins were mixed with 20X E1 enzyme, 10X Mg²⁺-ATP solution, 10X ubiquitin solution, 1 μg E2 enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer. The reaction was carried out at 37 °C for 1 h, and the products were analyzed by western blotting with an anti-P53 antibody (Santa Cruz, SC126).