Ric3 pcmv6 xl5

Mouse neuroblastoma Neuro2a cells were purchased from the Russian collection of cell cultures (Institute of Cytology, Russian Academy of Sciences, Saint Petersburg, Russia). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Paneco, Russia) supplemented with 10% fetal bovine serum (PAA Laboratories, Austria). They were sub-cultured the day before transfection and were plated at a density of 10000 cells per well (96-well plate) or 50000 cells per poly-D-lysine-coated glass coverslip (12 mm in diameter). On the next day Neuro2a cells were transiently transfected with plasmids coding α7 nAChR (human α7 nAChR-pCEP4, rat α7 nAChR-pcDNA 3.1/Hygro(+)) or its mutants, the chaperone Ric-3 (Ric3-pCMV6-XL5, OriGene, USA) or NACHO (TMEM35-pCMV6-XL5, OriGene, USA) and a fluorescent calcium sensor Case12 (pCase12-cyto vector, Evrogen, Russia) in molar ratio 4:1:1 following a lipofectamine transfection protocol (Invitrogen, USA). Wild-type or mutant mouse muscle α1β1δε nAChRs (pRBG4-vector) were expressed accordingly, not requiring a chaperone. The transfected cells were grown at 37°C in a CO₂ incubator for 48–72 h, before binding and function were assessed.

Mouse neuroblastoma Neuro2a cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Paneco, Russia) supplemented with 10% FBS (PAA Laboratories, Austria). Cells were sub-cultured 24 h before transfection and plated at a density of 10,000 cells per well (black 96-well plate, Corning, USA), followed by lipofectamine (Invitrogen, USA)-mediated transient co-transfection of human α7 nAChR-pCEP4, fluorescent calcium sensor pCase12-cyto (Evrogen, Russia) and chaperone Ric3-pCMV6-XL5 or NACHO TMEM35-pCMV6-XL5 plasmid constructs (OriGene, USA). Mouse muscle α1, β1, ε, and δ nAChR-pRBG4 plasmid constructs were expressed similarly, but without a chaperone, as well as mouse α1, β3, γ2 GABA_AR-lab-pCI plasmids.