FIGURE 10.
Tariquidar stabilizes TMD1. A, A52-tagged TMD1 (T1) (residues 1–379), A52-tagged TMD2 (T2) (residues 681–1025), or both A52-tagged TMD1 plus A52-tagged TMD2 (T1 + T2) were expressed in the absence (−) or presence (+) of 1 μm tariquidar (Tar). Whole cell SDS extracts were subjected to immunoblot analysis. The locations of TMD2 (T2) and mature and immature forms of TMD1 (T1) are indicated. B, the amount of mature TMD1 (T1) relative to total (mature and immature TMD1) (% Mature T1) was quantified. Each value is the mean ± S.D. (n = 3–5). An asterisk indicates a significant difference (p < 0.001) relative to samples grown in the absence of tariquidar. C, HEK 293 cells expressing A52-tagged TMD1 plus untagged TMD2 and grown in the presence of tariquidar were treated without (−) or with (+) endoglycosidase H (Endo Hf) or PNGase F. The reactions were stopped by addition of SDS sample buffer and subjected to immunoblot analysis. The positions of mature, immature, and unglycosylated (Unglycos) forms of TMD1 are indicated. D, membranes prepared from cells expressing A52-tagged TMD1 plus untagged TMD2 and grown in the absence (None) or presence (+Tariquidar) of tariquidar were treated with various concentrations of trypsin. The reactions were stopped by addition of trypsin inhibitor and samples were subjected to immunoblot analysis. The positions of mature and immature forms of TMD1 are indicated. E, the levels of mature (filled bars) or immature (unfilled bars) forms of TMD1 were determined and the amount remaining (% Remaining) after treatment with various levels of trypsin was compared with the untreated control. An asterisk indicates a significant difference (p < 0.001; n = 3) relative to that without trypsin.