thylrhodamine. 50 nM labeled reporter peptide was mixed with MAPKs in a concentration PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844759 to achieve ~6080% complex formation. Subsequently, increasing amounts of chemically synthesized test peptides were added, and the FP signal was measured with a Synergy H4 plate reader in 384-well plates. The Ki for each unlabeled peptide was determined by fitting the data to a competition binding equation. Titration experiments were carried out in triplicates, and the average FP signal was used for fitting the data with OriginPro7. Cell-based proteinprotein interaction assay The full-length cDNA of yellow fluorescent protein was split at position 159, and fragments were pasted into pcDNA 3.1 vectors. ERK2 was expressed as C-terminal and p38a and JNK1 as N-terminal F2 fusions. To facilitate expression, JNK1 and p38a had kinase-inactivating mutations, while ERK2 was wild type. The ERK2 and JNK1 constructs contained a FLAG-tag, but similarly tagged p38a constructs could not be expressed to a comparable extent. Therefore, expression levels of F2-p38a could only be monitored by an anti-p38a antibody. MAPK partners were expressed as N-terminal and C-terminal F1 fusions with FLAG-tags. F1 and F2 fusion pairs that gave the highest BiFc signal with wild-type MAPK partners were chosen to analyze the impact of docking motif truncations or mutations. These were introduced into full-length MAPK partner constructs by PCR or by the QuickChange method. N-terminal truncations were made in proteins with N-terminal docking motifs: MKK1, MKK6, RHDF1, MKP5, and FAM122A and a 20-amino-acid-long C-terminal truncation was used to generate the DCX construct. Mutation of multiple residues was utilized for proteins with internal motifs: JIP1 and APBA2, and K331, K332, K333, and L337 were mutated to alanines in the short KSR2 construct All sequences were verified by DNA sequencing. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37C in an atmosphere of 5% CO2 in 25-cm2 tissue culture flasks. Cells were seeded onto 96-well plate at 6070% confluence 24 h prior to transfection. The medium was changed to serum-free OPTI-MEM. Transient transfections with Lipofectamine 2000 reagent were carried out according to the manufacturer’s instruction. Cells were assayed 2 days post-transfection. For BiFc signal intensity measurements, cells were washed and suspended in 100 ll PBS. Twenty microliters of this cell suspension was aliquoted into a 384-well black-sided plate. Fluorescence intensity per well was measured using a Synergy H4 fluorescence plate reader. Cells from 50 ll of the PBS suspension were collected, and samples were subjected to Western blots using anti-FLAG-tag antibody. For imaging, transfected cells were examined with an Olympus IX81 microscope using an Olympus FluoView 500 confocal laser scanning microscope system. YFP fluorescence was imaged using 514-nm excitation and a 535- to 560-nm emission filter. Cell-based MedChemExpress A-83-01 assays for EGF stimulation and monitoring GAB1 localization GAB1 constructs were subcloned into modified pCerulean-C1 vector with N-terminal Cerulean fluorescent protein and C-terminal FLAG fusion tags. HEK293T cells were cultured and transfected similarly as described above. Cells were transfected with 0.2 lg GAB1 DNA constructs and 
were serum-starved for 24 h. The media were removed after 40 h from DNA transfection and 100 ll PBS was added to wells. ERK pathway stimulation was st