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. 2011 Jul 13;286(39):33811–33818. doi: 10.1074/jbc.M111.270207

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — The effect of SOCS1 on Tyk2 ubiquitination. Western blots analyses (WB) and immunoprecipitation (IP) Western blot analyses of Tyk2 ubiquitination. A, Tyk2-HIS was expressed in HEK293T cells with ubiquitin-HA, ubiquitin(K63)-HA, or ubiquitin(K48)-HA ± SOCS1-FLAG or SOCS1Δ170–212 in the presence of MG132. Cell lysates were immunoprecipitated with nickel affinity beads. Immunoprecipitates and whole cell lysates (WCL) were separated by SDS-PAGE and probed with the indicated antibodies. The second panel from the top shows a shorter exposure than the top panel. Tyk2 ubiquitination is shown in the top panel, third lane, and Lys-63 ubiquitination in the top panel and the second panel from the top, fifth lane. Inhibition by SOCS1 constructs is evident in the fourth lane (top panel) and the sixth and seventh lanes (top panel or second panel from the top). B, Tyk2KD-GST or Tyk2KD(2F)-GST were expressed in HEK293T cells ± ubiquitin-HA. Cell lysates were immunoprecipitated with glutathione beads. Immunoprecipitates were separated by SDS-PAGE and probed with the indicated antibodies. Blots are representative of triplicate experiments.