Journal: Nature

Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

doi: 10.1038/nature18268

Figure Lengend Snippet: Cx43 directly interacts with PCDH7, but not with E cadherin or N cadherin a, Schema illustrating split luciferase assay. Fusion constructs of PCDH7 and Cx43 were created with either the N or C terminal halves of luciferase, NLuc and CLuc, respectively. When these proteins are brought into proximity, luciferase is functionally reconstituted, producing photons of light. b, Cx43 and PCDH7 constructs fused with N-terminal and C-terminal firefly luciferase halves (N-luc) and (C-luc) were expressed in parental cell lines. The table (top) numerically identifies the cell line combinations used in the assays (bottom), and bioluminescence imaging (BLI) of a representative plate. c , Cx43 and PCDH7 western immunoblotting in cancer cells overexpressing fusion proteins. d, Quantification of BLI after co-culture of Cx43-CLuc/PCDH7-NLuc(+) cancer cells and astrocytes for 15 min. (3 independent experiments) e-g, Luciferase split assay to detect Cx43-E cadherin or Cx43-N cadherin interactions. Cell line combinations used in the assays are numerically identified in the table ( e ), and confirmed by western immunoblotting ( f ). Bioluminescence imaging (BLI) of a representative assay plate; cell line combinations are indicated numerically ( g ). (n ≥ 2 independent experiments).

Article Snippet: Split luciferase assay Fusion cDNAs were generated by deleting the stop codon in human Cx43 (Origene), PCDH7 (Origene), E-cadherin (Addgene) or N-cadherin (Addgene) cDNAs and splicing the N-terminal or C-terminal fragment of firefly luciferase . (Addgene).

Techniques: Luciferase, Construct, Imaging, Western Blot, Co-Culture Assay