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. 2017 Oct 25;6:e28766. doi: 10.7554/eLife.28766

Figure 6. RNF145 ubiquitinates SCAP to inhibit SREBP processing.

(A) CHO cells were transfected with plasmids encoding HSV-tagged SCAP and HA-tagged ubiquitin, together with RNF145 or RING-mutant RNF145 (C537A). Cell lysates were blotted with the indicated antibodies. Cell lysates were also immunoprecipitated with HSV antibody, and the precipitated proteins were blotted with HA antibody. (B) HEK293T cells were transfected with plasmids encoding HSV-tagged SREBP-2 under the control of a TK promoter, together with GFP control, RNF145 or RING-mutant RNF145, GP78, or TRC8. Cells were cultured in DMEM medium supplemented with 10% FBS, or in DMEM medium supplemented with 10% lipoprotein-deficient serum and 25 μM simvastation overnight to deplete sterol, and then treated for 4 hr with MG132 (25 μM). Membrane and nuclear protein fractions of the cells were then isolated and blotted with the indicated antibodies. (C) CHO cells were transfected with plasmids encoding RNF145 and HA-tagged ubiquitin, together with HSV-tagged SCAP or mutant SCAP (K454R, K466R). Cell lysates were blotted with the indicated antibodies. Cell lysates were also immunoprecipitated with HSV antibody, and the precipitated proteins were blotted with HA antibody. (D) SCAP-knockout HEK293 cells were transfected with plasmids encoding HSV-tagged SREBP-2 under the control of a TK promoter and RNF145, together with V5-tagged SCAP or ubiquitination-site mutant SCAP (K454R, K466R). Cells were cultured in DMEM medium supplemented with 10% lipoprotein-deficient serum and 25 μM simvastation overnight to deplete sterol, and then treated for 4 hr with MG132 (25 μM). Membrane and nuclear protein fractions were isolated and blotted with the indicated antibodies.

Figure 6.

Figure 6—figure supplement 1. Interaction between RNF145 and the SCAP pathway.

Figure 6—figure supplement 1.

(A) Immunoblot analysis of CHO cells transfected with plasmids encoding HSV-tagged SCAP and FLAG-RNF145. Cell lysates were blotted with the indicated antibodies. Cell lysates were also immunoprecipitated with HSV antibody, and the precipitated proteins were blotted with FLAG antibody. (B) Immunoblot analysis of CHO cells transfected with plasmids encoding HSV-tagged SCAP and FLAG-RNF145 and treated with cycloheximide (10 µg/ml) as indicated. (C) Quantification of the immunoblot signals from Figure 6B. (D) Schematic diagram of the SCAP protein, showing the relative location of the lysine residues ubiquitinated by RNF145 (yellow) and the COPII-binding amino acid sequence (red) within cytoplasmic loop 6 of SCAP. (E) Real-time PCR analysis of gene expression in mice transduced with RNF145 for the indicated times. Gene expression normalized to expression levels in GFP-transduced controls.