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. 2017 Jan 9;36(4):425–440. doi: 10.15252/embj.201694314

Figure 6. Conservation of ubiquitylation‐dependent oligomerization in human Nedd4 and implications in targeting of FGFR1.

Figure 6

  1. Elution profile from size‐exclusion chromatography of apo HECT (α1Nedd4) or Ub‐fused HECT (α1Nedd4‐Ub) of Nedd4. Proteins were loaded on a Superdex 200 16/60 column, and elution was monitored by A280 measurements.
  2. In vitro ubiquitylation of Rpn10 by apo HECT (α1Nedd4) or Ub‐fused HECT (α1Nedd4‐Ub) of Nedd4. Reaction products were resolved by SDS–PAGE and blotted against a primary rabbit α‐Rpn10 antibody, followed by secondary IR labelled mouse α‐rabbit. Odyssey infrared imaging system was used for IR detection.
  3. Quantified ubiquitylated/total Rpn10 ratio (mean values and standard deviation bars from three replicates).
  4. Documentation of Nedd4 knockdown by shRNA in HeLa cells.
  5. Representative immunoblot of FGFR1 ubiquitylation (in the presence of serum) upon transfection of the indicated wild‐type (WT) and mutant human Nedd4 constructs. V5‐Nedd4 was used as an additional control.
  6. Quantified (mean ± SEM) ubiquitylated/total FGFR1 ratio from three separate experiments. P‐values are from Student's t‐test.