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Fig. 8. (A) Generation of a SOCS2 mutant deficient in elongin B/C binding. HEK293T cells were transiently transfected with the pMET7TAP2-SOCS2 and pMET7TAP2-SOCS2(LC-QQ) constructs. Cell lysates were purified using the TAP2 tag and loaded on a polyacrylamide gel and silverstained. From a parallel experiment, the indicated bands were identified as cullin 5, elongin B and elongin C by mass spectrometry. (B) The SOCS2(LC-QQ) mutant still binds CIS. HEK293T cells were transiently co-transfected with plasmids encoding the chimeric EpoR-LR(F3) construct as a negative control or with the CIS SOCS box bait, and the pMG2-SOCS2 or pMG2-SOCS2 (LC-QQ) prey constructs, combined with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with Epo or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (Epo stimulated/NS) + s.d. (C) Differential effects of the SOCS2(LC-QQ) mutant on CIS interaction with the LR recruitment motifs. HEK293T cells were transiently co-transfected with plasmids encoding different pMet7-LR variants, the pMG2-CIS prey construct, pMet7-FLAG-SOCS2 or pMet7-FLAG-SOCS2(LC-QQ), or the appropriate amount of mock vector together with the pXP2d2-rPAP1-luci. The transfected cells were either stimulated for 24 hours with leptin or were left untreated (NS, not stimulated). Luciferase measurements were performed in triplicate. Data are expressed as mean fold induction (leptin stimulated/NS) + s.d.

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