Wang, Yong-Jian
Bian, Yan
Luo, Jie
Lu, Ming
Xiong, Ying
Guo, Shu-Yuan
Yin, Hui-Yong http://orcid.org/0000-0001-7049-1560
Lin, Xu
Li, Qin
Chang, Catherine C. Y.
Chang, Ta-Yuan
Li, Bo-Liang http://orcid.org/0000-0002-7123-5638
Song, Bao-Liang http://orcid.org/0000-0002-6397-5935
Article History
Received: 22 December 2016
Accepted: 10 May 2017
First Online: 12 June 2017
Change Date: 13 November 2017
Change Type: Corrigendum
Change Details: In the original version of this Article, the procedure for assessing the ubiquitylation assays by western blotting was inadvertently omitted from the Methods. This method was used in Figures 2b,d,g,h, 3a,b,d, 4f and 5h and Supplementary Figures 3b, 5b and 6b, and was designed to omit reagents such as BME and DTT that might break the thioester bond. The full method is given below, and has been added to the Methods section of the Article. Ubiquitylation assay western blot. For Figs 2b,d,g,h, 3a,b, 4f and 5h, and Supplementary Figs 5b and 6b, after the last wash of the beads, the supernatant was discarded and beads were boiled for 10 min in 100 μl 2× SDS loading buffer (75 mM Tris-HCl, pH 6.8, 6% SDS, 15% (v/v) glycerol, 0.01% (w/v) Bromophenol blue) and were then vortexed and centrifuged at 1,000 g for 2 min. 90 μl supernatant was mixed with 90 μl solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M Urea, 10% glycerol) and incubated at 37 °C for 30 min. For Fig. 3d and Supplementary Fig. 3b, the beads were incubated with 100 μl Myc peptide (1 mg/ml) for 3 hrs at 4 °C, vortexed and centrifuged at 1,000 g for 2 min. 90 μl supernatant was mixed with 90 μl solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M Urea, 10% glycerol) and 60 μl modified 4× SDS loading buffer (150 mM Tris-HCl, pH 6.8, 12% SDS, 30% (v/v) glycerol, 0.02% (w/v) Bromophenol blue) and incubated at 37 °C for 30 min. Samples were resolved by SDS–PAGE and transferred onto PVDF membranes. Immunoblots were blocked with 5% BSA in TBS containing 0.075% Tween (TBST) and probed with primary antibodies overnight at 4 °C. After washing in TBST 3 times, blots were incubated with secondary antibodies for 1 h at room temperature. After washing in TBST 3 times, bands were visualized by enhanced chemiluminescence (ECL).
Competing interests
: The authors declare no competing financial interests.