Pcibn deltanls pmgfp

Cloning of I-BAR–Cry2–mCh, Cry2–mCh–I-BAR, I-BAR–Cry2–mCh–WH2, and Cry2–mCh–MTSS1 constructs was conducted using a previously described cloning scheme (34 (link)). Briefly, the I-BAR domain from MTSS1, the WH2 domain from MTSS1, and MTSS1 were PCR amplified from human MTSS1 complementary DNA obtained from the Arizona State University DNA repository (DNASU ID: HsCD00746054). N-terminal I-BAR genes were cloned into Cry2PHR–mCherry (Addgene; #26866) using NheI and XhoI restriction sites (23 (link)). C-terminal I-BAR, WH2, and MTSS1 genes were cloned into Cry2PHR–mCherry using BsrgI and NotI restriction sites. The ezrin–GFP expression construct was a gift from Stephen Shaw (plasmid pHJ421; Addgene; #20680 (35 (link))). The actin expression construct (pCAG-mGFP-actin; Addgene; #21948) was a gift from Ryohei Yasuda. CIBN–GFP–CAAX (pCIBN(deltaNLS)-pmGFP; Addgene; #26867) was a gift from Chandra Tucker. Midi prep quantities of DNA of each construct were created from Escherichia coli and collected for cell transfection.

For mammalian expression of the LuminON system, the coding sequence of Nanoluc was cloned into pGAVPO and pSV40-GAVPO^{27 (link)} using the Hieff Clone® Plus One Step Cloning Kit (YEASEN) to obtain plasmids expressing different configurations of Nluc-GAVPO fusion proteins driven by the CMV or SV40 promoter. For application of the LOVTRAP or CRY2-CIB1 system, Nanoluc was ligated into the pTriEx-mCherry-LOV2 (Addgene: 81041) plasmid digested by Acc65I and NheI (Thermo Scientific), the pCIBN(deltaNLS)-pmGFP (Addgene: 26867) plasmid digested by Eco47III and AgeI (Thermo Scientific), or the pCRY2FL(deltaNLS)-mCherryN1 (Addgene: 26871) plasmid digested by Eco47III and XhoI (Thermo Scientific). For cell therapy of the LuminON system in mice, luminGAVPO was ligated into the pYH88 plasmid digested by Eco47III and BsrGI, and UAS_G-TATA-Gluc-P2A-mINS was ligated into the pWS251 (obtained from Haifeng Ye, ECNU) plasmid digested by NotI and MluI.