origin 7 software package  (Malvern Panalytical)

Journal: Cellular and Molecular Life Sciences

Article Title: PSTPIP1-LYP phosphatase interaction: structural basis and implications for autoinflammatory disorders

doi: 10.1007/s00018-022-04173-w

Figure Lengend Snippet: Interaction of LYP with F-BAR dimers. A , B HEK293 cells were transfected with PSTPIP1 F-BAR dimers in 3xmyc plasmid, as indicated in the top of the panels, and with 3xFLAG-LYP. Lysates were immunoprecipitated with myc Ab and LYP bound to PSTPIP1 was detected by IB with Ab for LYP. Expression of the proteins was verified by IB in total lysates (TL) with the same Abs. C Determination of the interaction stoichiometry by titration of fluorescein-LYP-CTH (1 µM) with the F-BAR of PSTPIP1 (residues 1–289) using fluorescence anisotropy. The stoichiometry ( N ) was determined from the intersection of asymptotic lines in the linear ascending and saturation (i.e. plateau) regions. D ITC analysis of LYP binding to PSTPIP1. Upper panel, heat exchange of the dilution of the unlabeled LYP-CTH peptide in buffer 20 mM sodium phosphate (pH 7.5), 150 mM NaCl, 5% (v/v) DMSO (upper trace) and representative thermogram of the binding of LYP-CTH to the F-BAR of PSTPIP1 (lower trace). Lower panel, LYP binding isotherm to PSTPIP1. The red line is the fit of a one binding site model to the heat changes of the interaction after subtracting the heat change of the dilution of LYP in buffer. The reported values of N , k d , and binding enthalpy (Δ H ) are the means ± standard deviations of three independent experiments

Article Snippet: Data were analyzed using the Origin 7 ITC software package (MicroCal Software, Northampton, MA, USA), corrected by the heat of injection calculated from the basal heat remaining after saturation and confirmed by titration into buffer only as control.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Titration, Fluorescence, Binding Assay