To rule out the possibility that nonspecific electrostatics dominates cNPF1 binding, we tested a cyclic peptide that maintained the negative charge but lacked the critical NPF motif (cAPAFlu).
To assess the influence of the N-terminal fluorescein dye on binding, we tested a subset of unlabeled peptides, MS1, MS2, Bim, and NoxaA in competition with a fluorescently labeled Bim variant (Supplementary Figure 2).
To map the residues in talin 2300 2541 directly or indirectly involved in actin binding, we tested the effects of a series of mutations on its affinity for F-actin.
To identify the protein region responsible for RNA binding, we tested PPARγ deletion mutants.
To confirm that this region is involved in RSK2 binding, we tested binding of RSK2 to three full-length GST-Tat fusion proteins with alanine-scanning mutations in the core region (Figure 3D, lanes 5, 6, 7).
Concerning DNA binding, we tested whether S/A or S/D mutation of the N- and C-terminal serines would have an effect on binding to the C-box consensus motif as it was shown for S160, 164, and 168 in the DNA binding domain (Kirchler et al., 2010, The role of phosphorylatable serine residues in the DNA-binding domain of Arabidopsis bZIP transcription factors).
To further define the sequence requirement for PhoP binding, we tested the effects of single-base substitutions on binding affinity.
To identify residues most important for binding, we tested additional deletions of the strongest site of interaction in the CRD.
To complement the in vitro binding assays, we tested a representative coiled-coil construct in mammalian cells and monitored its ability to interact with endogenous IKKβ.
We present two approaches to extract the binding relevant information: first we use visual inspection of a known ligand as well as literature review to identify binding relevant substructures, second we test a relatively basic data mining approach.
To confirm that mutants M-1-2, M-1, and M-2 lost their polyubiquitin binding activity, we tested their binding activity in an ELISA binding assay in which polyubiquitin was adsorbed on a microtitre plate.
