Figure 2: CSN6 regulates the SCF (Skp1/Cullin-1/Fbxw7) complex to stabilize Myc.

(a) CSN6 associated with Myc, Fbxw7 and Cullin-1. The U2OS cell lysates were immunoprecipitated with anti-CSN6 and immunoblotted with indicated antibodies. (b) CSN6 knockdown led to decreased Cullin-1 neddylation. The U2OS cell lysates were immunoprecipitated with anti-Cullin-1, followed by immunoblotting with indicated antibodies. (c) CSN6 expression was inversely related to Fbxw7 expression. Immunoblot analysis of Myc and Fbxw7α in CSN6-overexpressing or CSN6 knockdown cells. Cell lysates were analysed by indicated antibodies. (d) Knockdown of CSN6 increased the expression of Fbxw7 and reduced Myc. Indicated cells were infected with lentivirus to express one of two independent CSN6 shRNAs (#1 and #2) or luciferase control shRNA. Cell lysates were immunoblotted with indicated antibodies. (e) CSN6 reduced steady-state expression of Fbxw7 to upregulate Myc. Indicated cells were transfected with increasing amounts of GFP-CSN6. Cell lysates were immunoblotted with indicated antibodies. Fbxw7α and Fbxw7β isoforms were identified. (f) Knockdown of CSN6 enhanced Myc ubiquitination. Indicated cells were infected with lentivirus to express one of two independent CSN6 shRNAs (#1 and #2) or luciferase control shRNA. The cells were then transfected with His-ubiquitin and treated with MG132 for 6 h before harvest. The ubiquitinated Myc proteins were pulled down using Ni-NTA-agarose beads and detected with anti-Myc antibody.