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. 2020 Jan 1;219(2):e201908182. doi: 10.1083/jcb.201908182

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© 2020 Chen et al.

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Figure 1. — RTN4 binds to the ER membrane J proteins B12, B14, and C18. (a) Total number of peptides corresponding to RTN3 and RTN4 identified by MS using the FLAG immunoprecipitated material derived from either Flp-In 293T-Rex parental cells or cells stably expressing 3xFLAG-B12 or 3xFLAG-C18. (b) 3XFLAG-B12, 3XFLAG-B14, or 3XFLAG-C18 was immunoprecipitated from induced Flp-In 293T-REx cells. Extracts from the parental cells were used as a negative control. Bound proteins were eluted by 3× FLAG peptide and subjected to SDS-PAGE, followed by immunoblotting using the indicated antibodies. The asterisks in panels b and c indicate an antibody heavy chain, and the number signs in panels b and c might be RTN4D. (c) Cell extracts from mock-infected (left panel) or SV40-infected (right panel) CV-1 cells were incubated with either anti-B12, anti-B14, or a control IgG antibody. The immunoprecipitated materials were subjected to SDS-PAGE, followed by immunoblotting using the indicated antibodies. (d) COS-7 cells were transfected with GFP-RTN3C, and the cell extracts were subjected to immunoprecipitation by anti-B12, anti-B14, or a control IgG antibody. The immunoprecipitated materials were subjected to SDS-PAGE, followed by immunoblotting using the indicated antibodies. Input represents 1% of the total sample used for the immunoprecipitation.