Molecular Tagging of Murine Testis Proteins

Open reading frames encoding C11orf94, Izumo1, SOF1, SPACA6, and ACE3 were amplified by PCR from murine testis cDNA and equipped with C-terminal 3xFLAG, (3x)myc, or HA tags, respectively. Coding sequences were inserted into the pcDNA3.1 hygro⁺ vector (Thermo Fisher Scientific) using Hind III and Xho I (C11orf94, SPACA6, and Izumo1) or Bam HI and Xho I (SOF1 and ACE3) restriction sites. For analysis of the membrane topology of C11orf94, an artificial NIS motif was added to the N or C terminus of the protein, respectively. Plasmids encoding NotchΔE-eGFP (53 (link)), mSPPL2c-myc, and mSPP-myc and the catalytically inactive mSPPL2c D457A variant (20 (link)) have been described previously. The mutations D395A and F455L were introduced into the SPPL2c-coding sequence by site-directed mutagenesis, and the resulting open reading frames were ligated into the pcDNA hygro⁺ expression vector using Bam HI and Not I sites. For propagation of plasmids, Escherichia coli XL1-Blue cells (Stratagene) were used.