The GW1-pHRed, pCMVdeltaR8.2 and pCMV-VSV-G vectors were purchased from Addgene (Cambridge, MA, USA). The pLV-SV4-puro lentiviral vector was obtained from Dr. Peter Chumakov, Cleveland Clinic (Cleveland, OH, USA). Lentiviral vectors encoding short-harpin RNA (shRNA) to IMPDH2 along with a non-silencing control vector were purchased from Sigma and described previously21 (link). The GEVAL_sensors were PCR-amplified from the bacterial expression vectors with the following primers and cloned into the pLVp lentiviral expression system.
The IMPDH2-shRNA resistant vector was created with the Q5 site-directed mutagenesis kit (New England BioLabs, NEB, Ipswich, MA, USA) from our previously generated IMPDH2 lentiviral expression vector24 (link), using the following primers and following the manufacturer's instructions.
The lentiviral infection protocol was described previously32 (link). All infected cells were briefly selected for resistance to puromycin and used in the described assays.
The IMPDH2-shRNA resistant vector was created with the Q5 site-directed mutagenesis kit (New England BioLabs, NEB, Ipswich, MA, USA) from our previously generated IMPDH2 lentiviral expression vector24 (link), using the following primers and following the manufacturer's instructions.
The lentiviral infection protocol was described previously32 (link). All infected cells were briefly selected for resistance to puromycin and used in the described assays.