Expression Constructs

All Prom1 expression constructs were generated by site-directed mutagenesis from pCS2-Prom1-YFP (a gift from N. Sasai, Nara Institute of Science and Technology). The base Prom1 construct used is the human S1 isoform (NCBI accession {"type":"entrez-protein","attrs":{"text":"NP_001139319.1","term_id":"224994189","term_text":"NP_001139319.1"}}NP_001139319.1)⁴⁸. Prom1-Strep was subcloned into pLV-EF1a vector (a gift from K. Hochedlinger, Massachusetts General Hospital) for lentiviral transduction. Human Pcdh21 isoform 1 (NCBI accession {"type":"entrez-protein","attrs":{"text":"NP_149091.1","term_id":"16933565","term_text":"NP_149091.1"}}NP_149091.1) was synthesized (GenScript) and cloned into a pCDNA3.1 vector (Thermo Fisher) for mammalian cell transfection. mTagBFP2 was subcloned from pBAD (Addgene #54572, a gift from Michael Davidson) into a pET28a vector with a C-terminal Strep tag for bacterial overexpression. Ttyh1 was expressed from a pLX304 vector after addition of C-terminal Strep and His tags to an existing construct (Addgene #161676, a gift from Mike McManus). To stably express fluorescently tagged Prom1 WT and W795R variants in HeLa cells for live cell imaging, Prom1-StayGold and Prom1[W795R]-StayGold sequences were synthesized (GenScript) and cloned into a P2555 vector (a gift from S. Jakobs, Max Planck Institute for Biophysical Chemistry) to yield constructs pAH18 and pAH20, respectively.