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. 2011 Oct 20;286(50):42937–42948. doi: 10.1074/jbc.M111.310920

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Association of SelK with Derlins, SelS, and p97. A, HEK 293 cells were metabolically labeled with ⁷⁵Se for 40 h. During the labeling procedure, the cells were either untreated or treated with 10 μg/ml tunicamycin (shown as Tm at the top of the panel) for 24 h. Digitonin lysates (Input) were subjected to IP with Derlin-1 and Derlin-2 antibodies. The selenoprotein pattern was visualized using a PhosphorImager system, and protein occurrence was analyzed by Western blotting (WB) with the indicated antibodies. Migration and molecular weights of SelS, SelK, and major endogenous selenoproteins, thioredoxin reductase 1 (TR1), and glutathione peroxidase 1 (GPx1) are indicated by arrows. B, lysates of HEK 293 cells labeled with ⁷⁵Se for 38 h were subjected to IP with SelS (lane 1), SelK (lane 2), and β-actin (lane 3) antibodies. Radioactivity pattern on the membrane was detected by a PhosphorImager system (upper panel). The same membrane was subjected to Western blotting with p97 antibody (lower panel). C, schematic representation of constructs coding for the HA-tagged mouse SelK or SelS used for co-transfection with the constructs coding for FLAG-tagged human Derlins. Sec-to-Cys and Sec-to-Stop mutants of HA-tagged mouse SelK/SelS are shown by abbreviations Cys and tr, respectively. D, HEK 293 cells were co-transfected in a 1:1 ratio with the constructs coding for HA-tagged Cys-containing (Cys) or truncated (tr) forms of mouse SelK and the constructs coding for three FLAG-tagged human Derlins. Lanes 1 and 2, HA-tagged SelK and FLAG-tagged Derlin-1; lanes 3 and 4, HA-tagged SelK and FLAG-tagged Derlin-2; lanes 5 and 6, HA-tagged SelK and FLAG-tagged Derlin-3b; lane 7, HA-tagged Cys-containing form of SelK (control); and lane 8, untransfected cells (control). The cells were lysed 40 h after transfection and subjected to IP with anti-HA agarose. The IP samples were analyzed by Western blotting with the indicated antibodies. An asterisk marks the position of FLAG-tagged Derlin-3b; two asterisks mark the position of FLAG-tagged Derlin-1 and -2. E, HEK 293 cells were subjected to the same procedure as in D with the only difference being that the constructs coding for the HA-tagged mouse SelS were used instead of the HA-tagged SelK.