FIGURE 2.

Assay of the xylosyltransferase activity of TMEM5. A, immunoprecipitation of the Myc-tagged proteins. Soluble Myc-tagged proteins in the culture supernatant were immunoprecipitated with anti-Myc-agarose (rabbit polyclonal) and subjected to Western blotting with an anti-Myc antibody (goat polyclonal). The migration positions of the molecular weight standards are shown on the left. B and C, assay for TMEM5 activity with the FKTN or FKRP product. After a 12-h reaction at 37 °C, the substrate and product were separated by HPLC, and the UV absorbance was detected at 215 nm (B); the [¹⁴C]Xyl-labeled product was detected via liquid scintillation counting (C). D and E, the MALDI-TOF MS spectra of the TMEM5 substrate (D) and its product (E) were prepared by the nonradioactive reaction (37 °C for 12 h). F, function of the TMEM5 mutant proteins. After a 12-h reaction at 37 °C, the substrate and product were separated by HPLC, and the [¹⁴C]Xyl-labeled product was detected.