Figure 2. Defective IL-21R subcellular distribution, N-linked glycosylation, and signal transduction in family A. (A) High-resolution APD imaging in HeLa cells expressing wild-type (wt, Arg201) or mutant (mut, Arg201Leu) C-terminal, eGFP-fused, IL-21R demonstrated distinct subcellular distribution. In contrast to the IL-21Rwt-eGFP, which was expressed at the plasma membrane (PM; arrowheads), the IL-21Rmut-eGFP protein was dispersed in the endoplasmic reticulum (ER; arrow heads) and did not traffic to the plasma membrane. Nuclei were stained using DRAQ5. (B) High-resolution APD imaging in HeLa cells expressing wild-type (wt, Arg201) or mutant (mut, Arg201Leu) IL-21R-eGFP illustrates incomplete colocalization of IL-21RArg201Leu with transiently expressed RFP-tagged JAK3. (C) FACS analysis gated on intact HeLa cells that were lentivirally engineered to overexpress either γc/IL-21Rwt-eGFP (Arg201) or γc/IL-21Rmut-eGFP (Arg201Leu). The top image reveals that IL-21-Atto647N ligand binding correlates with the magnitude of eGFP expression in HeLa cells expressing γc/IL-21Rwt-eGFP (Arg201), but not in γc/IL-21Rmut-eGFP (Arg201Leu)–transduced HeLa cells. The bottom image demonstrates that IL-21R surface staining correlates with the eGFP expression in γc/IL-21Rwt-eGFP (Arg201)–transduced HeLa cells, whereas HeLa cells expressing γc/IL-21Rmut-eGFP (Arg201Leu) showed aberrant IL-21R surface expression. (D) Western blot analysis of IL-21R expression in EBV-BCL from P2 in comparison to healthy donors. Protein lysates were treated with PNGaseF (top; 60 min, 30 IU/µl) or Endo H (bottom; 60 min, 100 IU/µl) before electrophoresis. Data shown are representative of three independent experiments. D, denaturation buffer; R, reaction buffer. (E) Western blot analysis of STAT signaling in EBV-BCL from P2 and healthy donor (HD) upon stimulation with indicated γc-related cytokines: IL-2 (100 ng/ml), IL-4 (100 ng/ml), IL-7 (100 ng/ml), IL-15 (10 ng/ml), and IL-21 (10 ng/ml). Please note that for detection of STAT3 phosphorylation (Tyr705, Y705) upon stimulation with IL-2, IL-4, IL-7, and IL-15, a longer exposure was used compared with stimulation with IL-21. Data are representative of two independent experiments, and defective STAT phosphorylation in P2 upon stimulation with IL-21 was confirmed in three additional experiments. (F) Western blot analysis of STAT3 phosphorylation (Tyr705, Y705) in IL-21–stimulated HeLa cells that were lentivirally transduced with γc along with either IL-21Rwt or IL-21Rmut. Data are representative of three independent experiments. (G) Lentiviral gene transfer of wild-type IL-21R in patient’s fibroblasts induces IL-21–mediated STAT3 signaling. Western blot analysis of STAT3 phosphorylation (Tyr705, Y705) in IL-21–stimulated dermal fibroblasts that were lentivirally transduced with wild-type or mutated IL-21R (Arg201Leu). Data depicted are representative of four independent experiments. GAPDH or actin were used as loading controls in D–G.
Figure 2.

Defective IL-21R subcellular distribution, N-linked glycosylation, and signal transduction in family A. (A) High-resolution APD imaging in HeLa cells expressing wild-type (wt, Arg201) or mutant (mut, Arg201Leu) C-terminal, eGFP-fused, IL-21R demonstrated distinct subcellular distribution. In contrast to the IL-21Rwt-eGFP, which was expressed at the plasma membrane (PM; arrowheads), the IL-21Rmut-eGFP protein was dispersed in the endoplasmic reticulum (ER; arrow heads) and did not traffic to the plasma membrane. Nuclei were stained using DRAQ5. (B) High-resolution APD imaging in HeLa cells expressing wild-type (wt, Arg201) or mutant (mut, Arg201Leu) IL-21R-eGFP illustrates incomplete colocalization of IL-21RArg201Leu with transiently expressed RFP-tagged JAK3. (C) FACS analysis gated on intact HeLa cells that were lentivirally engineered to overexpress either γc/IL-21Rwt-eGFP (Arg201) or γc/IL-21Rmut-eGFP (Arg201Leu). The top image reveals that IL-21-Atto647N ligand binding correlates with the magnitude of eGFP expression in HeLa cells expressing γc/IL-21Rwt-eGFP (Arg201), but not in γc/IL-21Rmut-eGFP (Arg201Leu)–transduced HeLa cells. The bottom image demonstrates that IL-21R surface staining correlates with the eGFP expression in γc/IL-21Rwt-eGFP (Arg201)–transduced HeLa cells, whereas HeLa cells expressing γc/IL-21Rmut-eGFP (Arg201Leu) showed aberrant IL-21R surface expression. (D) Western blot analysis of IL-21R expression in EBV-BCL from P2 in comparison to healthy donors. Protein lysates were treated with PNGaseF (top; 60 min, 30 IU/µl) or Endo H (bottom; 60 min, 100 IU/µl) before electrophoresis. Data shown are representative of three independent experiments. D, denaturation buffer; R, reaction buffer. (E) Western blot analysis of STAT signaling in EBV-BCL from P2 and healthy donor (HD) upon stimulation with indicated γc-related cytokines: IL-2 (100 ng/ml), IL-4 (100 ng/ml), IL-7 (100 ng/ml), IL-15 (10 ng/ml), and IL-21 (10 ng/ml). Please note that for detection of STAT3 phosphorylation (Tyr705, Y705) upon stimulation with IL-2, IL-4, IL-7, and IL-15, a longer exposure was used compared with stimulation with IL-21. Data are representative of two independent experiments, and defective STAT phosphorylation in P2 upon stimulation with IL-21 was confirmed in three additional experiments. (F) Western blot analysis of STAT3 phosphorylation (Tyr705, Y705) in IL-21–stimulated HeLa cells that were lentivirally transduced with γc along with either IL-21Rwt or IL-21Rmut. Data are representative of three independent experiments. (G) Lentiviral gene transfer of wild-type IL-21R in patient’s fibroblasts induces IL-21–mediated STAT3 signaling. Western blot analysis of STAT3 phosphorylation (Tyr705, Y705) in IL-21–stimulated dermal fibroblasts that were lentivirally transduced with wild-type or mutated IL-21R (Arg201Leu). Data depicted are representative of four independent experiments. GAPDH or actin were used as loading controls in D–G.

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