Skip to main content
. 2010 Aug 1;21(15):2797–2807. doi: 10.1091/mbc.E10-03-0240

Figure 2.

Figure 2.

Biological activity of IL-15 and IL-15Rα-sushi gp130, WSX-1, LIFR, OSMR, and GPL chimeric receptor proteins. (A) Equal numbers of Ba/F3-gp130 cells stably transduced with IL-15-gp130, sushi-gp130, IL-15-WSX-1, sushi-WSX-1, IL-15-gp130 + sushi-gp130, IL-15-gp130 + sushi-WSX-1, and IL-15-WSX-1 + sushi-gp130 were cultured for 3 d in the absence of Hyper-IL-6. As a control Ba/F3-gp130 were cultured in the presence or absence of 10 ng/ml Hyper-IL-6 or IL-27. Proliferation was measured as indicated in Materials and Methods. (B) Detection of IL-15-gp130 homodimers by coimmunoprecipitation. COS-7 cells were transiently transfected with p409-IL-15-gp130 or p409-IL-15-gp130 and pEYFP-IL-15-gp130. Forty-eight hours after transfection cells were lysed and IL-15-gp130-EYFP was immunoprecipitated with anti-GFP mAbs. As a control lysates were incubated only with protein G agarose. Input, immunoprecipitation supernatant and protein G agarose control supernatant were separated by SDS-PAGE. Proteins were detected with anti-GFP mAbs and visualized by ECL detection. The membrane was stripped and probed with anti-gp130 Abs, and proteins were visualized by ECL detection. (C) Equal numbers of Ba/F3-gp130 cells stably transduced with IL-15-LIFR, IL-15-LIFR + sushi-gp130, IL-15-OSMR, IL-15-OSMR + sushi-gp130, sushi-GPL, and IL-15-OSMR + sushi-GPL were cultured for 3 d in the absence of Hyper-IL-6. Proliferation was measured as indicated in Materials and Methods. (D) Detection of heterodimeric interaction between IL-15-WSX-1 and sushi-gp130 by coimmunoprecipitation. COS-7 cells were transiently transfected with p409-IL-15-WSX-1 or p409-IL-15-WSX-1 and p409-sushi-gp130. Forty-eight hours after transfection cells were lysed and sushi-gp130 was immunoprecipitated with anti-gp130 Abs. As a control lysates were incubated only with protein G agarose. Input, immunoprecipitation supernatant and protein A agarose control supernatant were separated by SDS-PAGE. Proteins were detected with anti-c-myc mAbs and visualized by ECL detection. The membrane was stripped and probed with IL-15-gp130 Abs, and proteins were visualized by ECL detection. Detection of sushi-gp130 homodimers by coimmunoprecipitation. (E) COS-7 cells were transiently transfected with p409-sushi-gp130 or p409-sushi-gp130 and pEYFP-sushi-gp130. Forty-eight hours after transfection cells were lysed and sushi-gp130-EYFP was immunoprecipitated with anti-GFP mAbs. As a control, lysates were incubated only with protein G agarose. Input, immunoprecipitation supernatant and protein G agarose control supernatant were separated by SDS-PAGE. Proteins were detected with anti-GFP mAbs and visualized by ECL detection. The membrane was stripped and probed with anti-gp130 Abs, and proteins were visualized by ECL detection. (F) Specificity of coimmunoprecipitation analysis. COS-7 cells were transiently transfected with pcDNA3-BACE and pEYFP-sushi-gp130. Forty-eight hours after transfection cells were lysed, and sushi-gp130-EYFP was immunoprecipitated with anti-GFP mAbs. As a control lysates were incubated with only protein G agarose. Input, immunoprecipitation supernatant and protein G agarose control supernatant were separated by SDS-PAGE. Proteins were detected with an anti-Myc-tag mAbs and visualized by ECL detection. (G) Tyrosine-phosphorylation of IL-15-gp130 and sushi-gp130. COS-7 cells were transiently transfected with p409-IL-15-gp130 or p409-sushi-gp130 or p409-IL-15-gp130 and p409-sushi-gp130 and starved for the last 20 h before lysis. Forty-eight hours after transfection, COS-7 cells were treated with pervanadate as described in Materials and Methods and lysed rapidly. The gp130 chimeric proteins were immunoprecipitated with anti-gp130 Abs. Immunoprecipitation supernatant was separated by SDS-PAGE. Proteins were transferred onto PVDF membrane and were detected with an anti-phosphotyrosine (PY) mAbs and visualized by ECL detection. To confirm immunoprecipitation of IL-15-gp130 and sushi-gp130, Western blots against IL-15 or c-myc were also performed. (H) Equal numbers of Ba/F3-gp130 cells stably transduced with IL-15-gp130, IL-15-gp130 + sushi-gp130, IL-15-gp130 + sushi-WSX-1, and IL-15-WSX-1 + sushi-gp130 were cultured for 3 d in the absence of Hyper-IL-6 and increasing amounts of the soluble sushi domain. Proliferation was measured as indicated in Materials and Methods.