In vitro–specific targeting of A20-Ig peptide ligands. (A) ELISA binding analysis of selected phages clones (left) or phage derived N-biotinylated synthetic peptides (middle) to purified A20-Ig. Peptide binding to polyclonal mouse immunoglobulins is also shown (right). Absorbance was calculated as the difference between OD_405nm and OD_620nm. Mean absorbance values ± SEMs of 4 independent experiments are shown; RU indicates relative units. The wild-type f88-4 phage, or a scrambled peptide (pCNT) was used as a control for the binding analysis of the selected phage clones or peptides, respectively. (B) Binding of FITC-conjugated peptides to A20 target cells by flow cytometry. Values are the mean fluorescent intensities (MFIs), representative of 2 independent experiments (n = 4). (C) Surface plasmon resonance analysis of the binding of the pA20-36 to A20-Ig. (D) Colocalization of FITC-conjugated pA20-36 peptide with the surface A20-Ig, as shown by confocal microscopy. Scale bar = 10 μm. In the diagram, the intensity profile for the pA20-36 and A20-Ig channel along a line scan through a representative cell is shown. Pictures were captured with a Leica TCS SP2 confocal microscope with a HCX PL APO 63.0×/1.40 oil UV objective (NA1.40) in glycerol and acquired with Leica Confocal Software Version 2.61. Image manipulation was performed with Adobe Photoshop CS.