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. 2017 Mar 14;292(18):7435–7451. doi: 10.1074/jbc.M117.783662

Figure 3.

Figure 3.

RNF11 suppresses SMURF2 but not SMURF1 polyubiquitylation in the cell. A, RNF11 differentially affects polyubiquitylation of SMURF Hect E3 ligase family members. Untagged wild-type RNF11 was co-transfected with His-tagged ubiquitin alone or with N-terminally FLAG-tagged SMURF1 or SMURF2 for 48 h in HEK293T cells. Cell lysates were then prepared under denaturing conditions and subjected to nickel-agarose purification. Ubiquitylation of SMURFs and RNF11 was detected by probing of Western blots with anti-FLAG or RNF11 antibody. Untransfected cells (lane 1) and cells transfected with His-ubiquitin alone (lane 2) were included as negative controls. Predicted molecular masses were as follows: α-tubulin, 50 kDa; FLAG-SMURF2, 87 kDa; FLAG-SMURF1, 86 kDa; RNF11, 17.4 kDa. *, mobility of unmodified RNF11. B, SMURF2 polyubiquitylation in the cell is not impacted by RNF11 silencing. Untagged wild-type RNF11 was co-transfected with His-tagged ubiquitin alone or with N-terminally FLAG-tagged SMURF2 for 48 h in HEK293T or HEK293T stably expressing a RNF11 short hairpin. Cell lysates were then prepared under denaturing conditions and subjected to nickel-agarose purification. Ubiquitylation of SMURF2 and RNF11 was detected by probing of Western blots with anti-FLAG or RNF11 antibody. Untransfected cells (lane 1) and cells transfected with His-ubiquitin alone (lane 2) were included as negative controls.