Figure 5. The mutant γ2-PTC subunit mRNA level was decreased by NMD, while the undegraded mRNA was translated to the immature γ2-PTC subunit with an ER glycosylation pattern. A, The mRNA level of the γ2-PTC subunit was increased after UPF1 knockdown (n = 6). RNA levels were evaluated by TaqMan quantitative real-time PCR as described in Materials and Methods. *p < 0.05, one-way ANOVA with Bonferroni's multiple-comparison test. B, The protein level of the γ2-PTC subunit was increased also after UPF1 knockdown (n = 4). **p < 0.01, one-way ANOVA Bonferroni's multiple-comparison test. Error bars indicate SEM. C, In HEK293T cells, the γ2-PTC subunit is a stable protein that was not secreted into the culture medium. HA-tagged wild-type γ2S subunit cDNA, γ2-PTC subunit cDNA, and pcDNA empty vector were expressed in HEK293T cells. Culture media of each cell and total cell lysate were both collected and incubated with HA beads to pull down HA-tagged proteins. Pull-down proteins were eluted with HA peptide, separated by SDS-PAGE, and blotted with HA antibodies. The experiment was repeated three times, and a representative gel was shown. D, The γ2-PTC subunit had an ER glycosylation pattern. HA-tagged wild-type γ2L and γ2-PTC subunits were expressed in HEK293T cells as either single subunits or were coexpressed with α1β2 subunits (Receptor). Total cell lysates from each condition were collected and digested with endoglycosidase Endo H or PNGase F. Digested and undigested proteins were blotted with HA antibodies. U, Undigested; H, Endo H digested; F, PNGase F digested. The experiment was repeated four times, and a representative gel was shown.