Fig. 2.

CSN6 interacts with FASN and attenuates ubiquitin-mediated degradation of FASN to enhance FASN protein stability. a Flag-CSN6 was transfected into DLD-1 cells. Cell lysates were immunoprecipitated with anti-Flag M2 agarose beads, and immunoprecipitates of anti-Flag-CSN6 were separated using SDS–PAGE and stained with Coomassie Brilliant Blue (left). The protein bands were retrieved and analyzed by mass spectrometry (right). The position of FASN and Flag-CSN6 are indicated by arrows. b Flag-CSN6 or Flag-FASN were transfected into HEK293T cells. Cell lysates were immunoprecipitated (IP) with anti-Flag M2 agarose beads and immunoblotted (IB) with the indicated antibodies (top). HCT 116 cell lysates were immunoprecipitated with anti-CSN6 antibodies followed by immunoblotting with the indicated antibodies (bottom). WCL, whole cell lysates. c Colocalization of FASN and CSN6 in cytoplasm of U2OS cells. U2OS cells expressing Flag-CSN6 were subjected to immunofluorescence analysis with anti-Flag-CSN6 (green) and anti-FASN (red) (top). Nuclei are stained with DAPI (blue). Scale bar, 10 μm. The colocalization coefficients between the indicated proteins were shown (bottom). d HCT-8 (top) and HCT 116 (bottom) cell proteins were extracted and fractionated by gel-filtration chromatography. Chromatographic eluant profiles and the eluted positions of proteins are shown. An equal volume from each chromatographic fraction was analyzed by western blotting with indicated antibodies. e CSN6 wild-type (wt), N-terminal (N-ter) or C-terminal (C-ter) was transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag M2 agarose beads and immunoblotted with the indicated antibodies. Light, immunoglobulin light chain. f Western blot analysis of FASN protein in cells infected with indicated lentiviruses (left). qRT-PCR analysis was performed to measure the mRNA expression of FASN (right). g, h HCT 116 (g) or DLD-1 (h) cells infected with indicated lentiviruses were treated with cycloheximide (CHX, 100 μg/mL) for the indicated time points. Cell lysates were immunoblotted with indicated antibodies (left). The relative abundance of remaining FASN protein was normalized to the t = 0 controls (right). i–k HEK293T cells were transfected with indicated plasmids. MG132 (20 μM) was added to the cells 6 h before they were harvested. The ubiquitinated FASN proteins were pulled down with anti-Flag M2 agarose beads (i, k) or anti-myc beads (j) and detected with an anti-HA antibody. l HEK293T cells were transfected with the indicated HA-tag CSN6 constructs together with Flag-FASN. Cell lysates were immunoblotted with the indicated antibodies. All values are expressed as means ± SD. ns not significant, ****P < 0.0001; as determined by two-sided Student’s t-test or one-way ANOVA (f)