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. 2016 Apr 22;17(6):842–857. doi: 10.15252/embr.201541402

Figure 3. ΔSS mutant form of IL‐2Rα as a model for mislocalized proteins.

Figure 3

  1. Schematic representation of the IL‐2Rα proteins used in this study. WT: wild type, ΔSS: signal sequence (SS)‐deleted mutant, and ΔSSΔTM: SS and transmembrane domain (TM)‐deleted mutant.
  2. IL‐2Rα ΔSS mislocalized to the cytosolic fraction. HeLa cells, expressing Flag‐tagged IL‐2Rα WT or ΔSS, were fractionated to the cytosolic (Cyt.) and membrane (Mem.) fractions. Tubulin was used as a cytoplasmic marker, while calnexin was used for the ER membrane fraction.
  3. IL‐2Rα ΔSS was not glycosylated. Flag‐tagged IL‐2Rα WT or ΔSS proteins were expressed in HeLa cells and immunoprecipitated with an anti‐Flag antibody. The precipitates were incubated with (+) or without (−) 10 units of the deglycosylation enzyme PNGase F for 2 h and subjected to Western blot analysis with an anti‐Flag antibody. Glycosylated and non‐glycosylated signals are indicated.
  4. The glycosylated form of IL‐2Rα WT is a stable protein, while mislocalized IL‐2Rα ΔSS is degraded rapidly via its TMD. A series of IL‐2Rα deletion proteins were expressed in HeLa cells and then chased with 20 μg/ml CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantification graph of the left blot signals, and data represent mean ± SD calculated from four independent experiments (n = 4).
  5. IL‐2Rα ΔSS is degraded in a proteasome‐dependent manner. HeLa cells expressing Flag‐tagged IL‐2Rα ΔSS were treated with 20 μg/ml CHX as well as 0.1% DMSO (DM.), 10 μM MG‐132 (MG.), 2 μM bortezomib (Bor.), 10 μM leupeptin (Leu.), 0.1 μM bafilomycin A1 (Baf.), or 10 μg/ml pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates the quantified data of the left blot signals presented as mean ± SD calculated from three independent experiments (n = 3).
  6. The Flag‐tagged IL‐2Rα ΔSS mutant and T7‐tagged ubiquitin were co‐expressed in HeLa cells, and the cells were treated with (+) or without (−) 10 μM MG‐132. After 4 h, Flag precipitates were blotted with anti‐T7 and anti‐Flag antibodies.
  7. HeLa cells expressing Flag‐tagged IL‐2Rα WT, ΔSS, and ΔSSΔTM proteins were treated with (+) or without (−) 10 μM MG‐132. At 4 h after MG‐132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non‐glycosylated (and thus defective) IL‐2Rα WT (indicated by the arrowhead) is detected strongly in the presence of MG‐132.

Source data are available online for this figure.