FIGURE 3.

NRX does not affect steady state expression of α4β2 AChRs. A, NRX does not affect steady state levels of α4β2 AChRs. Whole cell lysates of tsA 201 cells expressing α4β2 and pEF6A vector only or coexpressing α4β2 and NRX were separated by SDS-PAGE and Western blotted with rat anti-α4 (mAb 299), goat polyclonal anti-β2 (C-20), or goat polyclonal anti-neurexin-1 (P-15). Duplicates of each condition are shown. B, NRX does not affect the surface expression of recombinant α4β2 AChRs in tsA 201 cells. tsA 201 cells were transfected with α4β2, α4β2+pEF6A vector, and α4β2+NRX. Cell surface expression of α4β2 was measured using an enzyme-linked immunoassay in which tsA 201 cells were washed, blocked, and then incubated with mAb (mAb 295). The cells were blocked again, fixed, and incubated with horseradish peroxidase-conjugated secondary Abs followed by incubation with the horseradish peroxidase substrate. The absorbance of the supernatant was then measured at 655 nm in a Beckman spectrophotometer. C, coexpression of NRX with α4β2 AChRs does not affect surface NRX expression. tsA 201 cells were transfected with NRX, NRX+pEF6A vector, and NRX+α4β2, and cell surface expression of NRX was measured using an enzyme-linked immunoassay using anti-VSV-G. The values in B and C are each from three separate experiments, expressed as the means ± S.E., and analyzed using analysis of variance test. The differences are not significant (p > 0.05).