Inducible Overexpression Cell Line Generation

For generating an inducible overexpression cell line, we used the T-REx™ System from Thermo Fisher Scientific. For Gateway cloning pT-REX-DEST30 vector was used as destination vector and pDEST-SH2D4A-myc-Flag was used as origin plasmid whose generation has been described before^{6 (link)}. First, HLF cells were transfected with pcDNA6/TR expressing the Tet repressor (TetR) gene under control of the human CMV promoter followed by antibiotic selection with 5 µg/ml blasticidin (InvivoGen, San Diego, CA, USA). In a second transfection step, the pT-Rex-DEST-30-SH2D4A-myc-Flag vector was introduced into HLF cells stably expressing the Tet repressor. Positive transfected cells were selected with geneticin (G-418; 800 µg/ml; G-418 disulfate salt solution; Sigma-Aldrich, Taufkirchen, Germany).