Fig. 3. Simultaneous phosphorylation of two 14-3-3 binding motifs pSer342 and pSer448 is required for tight complex formation between pNedd4-2^335–455 and 14-3-3η.

a 12% Native TBE-PAGE showing the interaction between 14-3-3η (240 pmol) and Nedd^335–455 variants without (no pS) or with one, two or three phosphorylation sites (120 pmol); 14-3-3η protein alone was loaded on the penultimate lane. b–i Sedimentation velocity analytical ultracentrifugation analysis of the complexes between 14-3-3η and pNedd4-2^335–455 variants showing the s_w isotherms of 14-3-3η and Nedd4-2 with all three phosphorylation sites pSer³⁴² + pThr³⁶⁷ + pSer⁴⁴⁸ (b), with no phosphorylation sites (c), with one phosphorylation site (pSer³⁴² (d), pThr³⁶⁷ (e) and pSer⁴⁴⁸ (f)), or with two phosphorylation sites (pSer³⁴² + pThr³⁶⁷ (g), pSer³⁴² + pSer⁴⁴⁸ (h) and pThr³⁶⁷ + pSer⁴⁴⁸ (i)). The isotherms of weight-averaged sedimentation coefficients were constructed by SV-AUC analysis of mixtures of 1 μM 14-3-3η with Nedd^335–455 variants (0.05 – 5 μM). The c(s) distributions underlying the s_w data points are shown in Supplementary Fig. S3.