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. 2017 Feb 28;13(5):900–913. doi: 10.1080/15548627.2017.1291479

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© 2017 The Author(s). Published with license by Taylor & Francis

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 6. — SPHK1 induces the lysine 63-linked ubiquitination of BECN1 through TRAF2. (A-C) SPHK1 induced the lysine 63-linked ubiquitination of BECN1 and did not affect the lysine 48-linked ubiquitination of BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with a plasmid encoding HA-BECN1. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-ubiquitin (A), anti-K63-specific ubiquitin (B) or anti-K48-specific ubiquitin (C). (D) TRAF2 was imperative for SPHK1 to induce the lysine 63-linked ubiquitination of BECN1. HepG2 cells were cotransfected with a plasmid encoding HA-BECN1 and control-siRNA (or si-TRAF2); then, the cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-K63-specific ubiquitin antibody. WT, wild type; Ub, ubiquitin; Con-siRNA, control-siRNA.