(
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.