Fig 1: Establishment of stable cell line expressing clustered AChR via a dual lentiviral system. (A) Schematic of our stable cell line expressing clustered AChR (KL525). It is established by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR a1, ß1, d, ? and the clustering protein rapsyn. (B) CMV-MCS-PGK-Puro lentiviral vectors expressing human rapsyn and the AChR a1 and ß1 subunits. (C) CMV-MCS-PGK-Blasticidin lentiviral vectors expressing the human AChR d and ? subunits. LTR, long terminal repeat; RRE, Rev Response Element; cPPT/CTS, central polypurine tract and the central termination; CMV, Cytomegalovirus; CHRNA1, Cholinergic Receptor Nicotinic Alpha 1 Subunit; CHRNB1, Cholinergic Receptor Nicotinic Beta 1 Subunit; CHRND, Cholinergic Receptor Nicotinic Delta Subunit; CHRNE, Cholinergic Receptor Nicotinic Epsilon Subunit; RAPSN, gene encoding rapsyn; PGK, phosphoglycerate kinase; WPRE, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element.
Fig 2: Stable expression of AChR subunits and rapsyn in HEK293T cells. (A) Double immunofluorescence staining with 4',6-diamidino-2-phenylindole (DAPI, blue), AChR subunits (red) and rapsyn (red) in the stable cell line KL525. Bar, 100 µm. (B–F) mRNA expression of the human AChR subunit genes CHRNA1, CHRNB, CHRND and CHRNE and the AChR-clustering protein gene RAPSN in KL525 cells, as measured by real-time PCR. Uninfected HEK293T cells were used as the negative control (NC). (G, H) Flow cytometric analysis and representative results of coexpression of the AChR a1 & d subunits in KL525 cells after passage. (I) Western blot analysis of AChR a1, ß1, d, ? subunits and rapsyn. Uninfected HEK293T cells were used as the NC. ****P < 0.0001 compared with NC.
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