Kinetic and affinity constants for CPP-adaptor binding to CBS-myoglobin (or TIP-myoglobin).

<p>Kinetic and affinity constants for CPP-adaptor binding to CBS-myoglobin (or TIP-myoglobin).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Cell penetration assay for a variety of CPPs and adaptor proteins for delivery of myoglobin to BHK cells.

<p>Each panel are images from experiments with different CPP-adaptors: A, TAT-CaM 2.0; B, CALML3; C, SAP-CaM; D, SAP(E)-CaM. BHK cells were treated for 1 h with DyLight 550 fluorescently labeled CBS-myo (rendered as white in left panels, red in center and right panels), in either the absence or presence of CPP-adaptor, washed and imaged live. Center images are optical sections set at a similar depth of the nucleus (NucBlue staining, white, center and right panels), as determined by position within the Z-stack. Orthogonal projections are shown at the right (boxed in red) and top (boxed in green) sides of each panel. Cytoplasmic compartments in live cells were visualized using CellTracker Green CMFDA dye (green in right panels). Comparison of CPP-adaptor-treated versus untreated cells indicates that in all cases, myoglobin was delivered and localized primarily to the cytoplasm. Scale bars in all panels, 20 μm.</p

TAT-CaM localization.

<p>Cell penetration assay performed with complexes of fluorescently labelled TAT-CaM and unlabeled myoglobin at 10 nM (top) and 100 nM (bottom). Fluorescence rendering is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178648#pone.0178648.g001" target="_blank">Fig 1</a> (Dylight fluorescence is white in the left panels and red in the center and right panels).</p

Biolayer interferometry analysis of CPP-adaptor-cargo binding.

<p>Association (0-300s) and dissociation (300-600s) phases are shown for A, TAT-CaM; B, TAT-CALL3; C, SAP-CaM; D, SAP(E)-CaM and E, TAT-Tropo binding to CBS-myoglobin (A-D) or TIP-myoglogbin. Analyte concentrations were 1000 nM (green), 500 nM (magenta), 250 nM (orange), 125 nM (red), and 63 nM (blue). Fits are shown to a single-state global model for which the constants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178648#pone.0178648.t001" target="_blank">Table 1</a>. F, EDTA dissociation phases for 1000 nM analyte samples moved into EDTA after dissociation phase. Binding normalized to % specific binding to eliminate differences in amplitude, allowing direct comparisons.</p

Biolayer interferometry analysis of subcellular localization constructs.

<p>TAT-CaM was used as ligand and analytes were CBS-myoglobin-NLS (green), CBS-myoglobin-KDEL (blue), CBS-myoglobin-SKL (red). Phases, analyte concentrations and fits are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178648#pone.0178648.g001" target="_blank">Fig 1</a>.</p

Cell penetration in myotubes.

<p>DyLight 550-labelled tubulin was used as cargo for TAT-CaM-mediated delivery to myotubes. Colors are rendered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178648#pone.0178648.g002" target="_blank">Fig 2</a>.</p