The SOCS2 Ubiquitin Ligase Complex Regulates Growth Hormone Receptor Levels
Figure 2
SOCS2 exhibits E3 activity in the presence of E1, E2, Ubiquitin and ATP.
(A) The FLAG-tagged SOCS2-Elongin B/C complex was immunoprecipitated from HEK293T cells transfected with FLAG-SOCS2 and Elongin B/C or Empty vector and Elongin B/C. In vitro ubiquitination assay was performed as described in Materials and Methods except for the following: for lane 1 the assay was performed with immunoprecipitate from cells transfected with empty vector instead of SOCS2, lane 3 lacks the E1 enzyme, lane 4 lacks the E2 enzyme, lane 5 lacks Ub, lane 6 lacks ATP. Western blots of the samples were performed as described. (B) Immunoprecipitate from cells transfected with mutant variants of SOCS2 was used for the In vitro ubiquitination assay. Assay was performed as described in the Materials and Methods. Cells were transfected with Elongin B/C and either Empty vector (lane 1), FLAG-SOCS2 (lane 2) or a mutant form of FLAG-SOCS2 (lanes 3 and 4) as specified in the picture. Western blots of the samples were performed as described.