Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) Binding isotherms of the IL-13 agonists displayed on the surface of yeast. Yeast-displayed IL-13 agonists were incubated with the indicated concentrations of biotinylated IL-13Rα1 ECD, stained with Alexa-647-coupled Streptavidin, and assessed via flow cytometry. Sigmoidal curves were fitted using Prism software (GraphPad). (B) Normalized KD binding affinities of the recombinant IL-13 agonists determined via surface plasmon resonance (SPR). The IL-13 KD was fixed to one and other agonists were normalized accordingly. (C) Summary of the amino acids mutated in the IL-13 agonists as well as the kon, koff and KD binding parameters values obtained from SPR experiments.

Article Snippet: Surface plasmon resonance SPR experiments were conducted on a Biacore T100 instrument using a Biacore SA sensor chip (GE Healthcare).

Techniques: Binding Assay, Incubation, Staining, Flow Cytometry, Software, Recombinant, SPR Assay

Journal: Molecular cell

Article Title: Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-induced Phospho-regulatory Circuit

doi: 10.1016/j.molcel.2016.10.029

Figure Lengend Snippet: (A) Biacore SPR analysis of in vitro RafRBD binding: K-Ras-GTP or K-Ras-GDP (10 µM) were injected over WT- or R89L-RafRBD and binding responses were determined. (B) A 2-fold dilution series of Rigosertib (50 µM to 0.39 µM) was injected over WT-RafRBD and binding was determined by SPR. (C) 5 µM WT-RafRBD was mixed with increasing concentrations of Rigosertib (0.19 – 50 µM), following which binding to avi-K-RasGTP was measured (red lines) and compared to a RafRBD calibration series (black lines) (D) WT- or R89L-GST-RafRBD was incubated for 1 hr with increasing concentrations of Rigosertib and binding to GTP-loaded WTor G12V-K-Ras was determined by proximity based energy transfer. As a control for nonspecific inhibition, the effect of Rigosertib on Avi-GST was also monitored. (E) HeLa cells were treated as indicated with DMSO or 2 µM Rigosertib (Rig) prior to stimulation with EGF for 5 min (+) and lysis. Raf dimerization was monitored by probing Raf immunoprecipitates for the presence of other Raf family members. Lysates were examined for activated pMEK, pERK, and pAKT levels and for total B-Raf, C-Raf, A-Raf and tubulin (loading control) levels. (F) Depiction of the Raf domain structure and location of the Rigosertib-induced sites of S/TP phosphorylation. (G) The indicated cancer lines were treated with DMSO (−) or Rigosertib (+) for 18 hrs prior to lysis. Endogenous C-Raf was immunoprecipitated and probed for pS642P levels or B-Raf. Shifts in the electrophoretic mobility of B-Raf and C-Raf were also examined.

Article Snippet: SPR binding experiments were performed on a Biacore S200 instrument (GE).

Techniques: In Vitro, Binding Assay, Injection, Incubation, Control, Inhibition, Lysis, Phospho-proteomics, Immunoprecipitation

Journal:

Article Title: Determination of the Adsorption Free Energy for Peptide-Surface Interactions by SPR Spectroscopy

doi: 10.1021/la8005772

Figure Lengend Snippet: Response curves (SPR signal (RU) vs time for TGTG-A-GTGT on CH3–SAMs at 25° showing an irreversible adsorption process even for this small peptide. The large drop in the SPR response when pure buffer was introduced at 90 s is primarily due to the bulk-shift effect.

Article Snippet: SPR Adsorption Experiments Adsorption experiments were conducted by SPR using a Biacore × instrument (Biacore, Inc., Piscataway, NJ).

Techniques: Adsorption