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. 2006 Jun;26(11):4063–4073. doi: 10.1128/MCB.01589-05

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Copyright © 2006, American Society for Microbiology

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FIG. 2. — Specificity of αJak2(PS523) and increased signaling activity by Jak2^S523A. A. HEK293 cells were cotransfected with ELR plus Jak2, made quiescent, and incubated for 15 min in the absence (−) or presence (+) of Epo (10 units/ml). Cells were lysed and immunoprecipitated with αJak2(758). Washed immunoprecipitates were either directly denatured (Regular) or were incubated with (With phosphatase) or without (No phosphatase) phosphatase for 30 min at 30°C. All proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblotting with the indicated antibody. Lower panel: the αJak2(NT) immunoblot was stripped and reprobed with an independently prepared αJak2(PS523)(CS). B. HEK293 cells were cotransfected with ELR plus control plasmid (pcDNA3) or the indicated Jak2 isoforms (Jak2 and Jak2^S523A), made quiescent, incubated in the absence (−) or presence (+) of Epo (10 units/ml) for 15 min, and lysed. Lysates or αJak2(758) immunoprecipitates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the indicated antibodies. The migration of detected proteins is noted to the right of the panels. C to F. Films corresponding to three independent experiments similar to that shown in panel B were quantified and the results plotted (means ± standard errors of the means). C and D. Tyrosine-phosphorylated Jak2 and Jak2 phosphorylated on Tyr_1007,1008, respectively, normalized to total Jak2. E and F. Tyrosine-phosphorylated STAT3 and phosphorylated ERK, respectively. G. HEK293 cells were cotransfected with ELR plus vector (V) or Jak2, made quiescent, and incubated for 2 h in the absence (−) or presence (+) of Epo (10 units/ml). Cells were lysed, and RNA was prepared and converted to cDNA for quantification of SOCS3 mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA. Relative expression was calculated by the 2^−ΔΔCt method; data are expressed as means ± standard errors of the means (n = 3). WT, wild type.

FIG. 2. — Specificity of αJak2(PS523) and increased signaling activity by Jak2^S523A. A. HEK293 cells were cotransfected with ELR plus Jak2, made quiescent, and incubated for 15 min in the absence (−) or presence (+) of Epo (10 units/ml). Cells were lysed and immunoprecipitated with αJak2(758). Washed immunoprecipitates were either directly denatured (Regular) or were incubated with (With phosphatase) or without (No phosphatase) phosphatase for 30 min at 30°C. All proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblotting with the indicated antibody. Lower panel: the αJak2(NT) immunoblot was stripped and reprobed with an independently prepared αJak2(PS523)(CS). B. HEK293 cells were cotransfected with ELR plus control plasmid (pcDNA3) or the indicated Jak2 isoforms (Jak2 and Jak2^S523A), made quiescent, incubated in the absence (−) or presence (+) of Epo (10 units/ml) for 15 min, and lysed. Lysates or αJak2(758) immunoprecipitates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the indicated antibodies. The migration of detected proteins is noted to the right of the panels. C to F. Films corresponding to three independent experiments similar to that shown in panel B were quantified and the results plotted (means ± standard errors of the means). C and D. Tyrosine-phosphorylated Jak2 and Jak2 phosphorylated on Tyr_1007,1008, respectively, normalized to total Jak2. E and F. Tyrosine-phosphorylated STAT3 and phosphorylated ERK, respectively. G. HEK293 cells were cotransfected with ELR plus vector (V) or Jak2, made quiescent, and incubated for 2 h in the absence (−) or presence (+) of Epo (10 units/ml). Cells were lysed, and RNA was prepared and converted to cDNA for quantification of SOCS3 mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA. Relative expression was calculated by the 2^−ΔΔCt method; data are expressed as means ± standard errors of the means (n = 3). WT, wild type.