Sequences encoding full-length (1-925) or N-terminal truncated (230–925 or 232–925) human RIG-I were cloned into either pcDNA5/FRT/TO (purchased from Thermo Fisher Scientific, Waltham, MA; for expression in human cells), pFBDM (for expression in insect cells) (Berger et al., 2004 (link)) or pETM11-SUMO3 (EMBL, Heidelberg, Germany; for expression in E. coli). All proteins that were overexpressed in human cells contained an N-terminal FLAG/HA-tag, whereas proteins purified from insect cells contained an N-terminal His-tag.
Mutants were generated by site-directed mutagenesis using the QuikChange protocol and PfuUltra polymerase (Agilent, Santa Clara, CA).
HEK293T RIG-I KO cells (Zhu et al., 2014 (link)) were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX, pyruvate and 10% fetal bovine serum (FBS) (all purchased from Thermo Fisher Scientific, Waltham, MA) at 37°C/ 5% CO2 and were regularly tested by PCR for potential mycoplasma contaminations. Spodoptera frugipeda Sf21 and Trichoplusia ni High Five insect cells were maintained at 27°C/ 150 rpm in SF-900 III serum-free medium and High Five serum-free medium supplemented with 10 mM L-glutamine, respectively (both purchased from Thermo Fisher Scientific, Waltham, MA).
Mutants were generated by site-directed mutagenesis using the QuikChange protocol and PfuUltra polymerase (Agilent, Santa Clara, CA).
HEK293T RIG-I KO cells (Zhu et al., 2014 (link)) were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX, pyruvate and 10% fetal bovine serum (FBS) (all purchased from Thermo Fisher Scientific, Waltham, MA) at 37°C/ 5% CO2 and were regularly tested by PCR for potential mycoplasma contaminations. Spodoptera frugipeda Sf21 and Trichoplusia ni High Five insect cells were maintained at 27°C/ 150 rpm in SF-900 III serum-free medium and High Five serum-free medium supplemented with 10 mM L-glutamine, respectively (both purchased from Thermo Fisher Scientific, Waltham, MA).
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