Figure 5. Nanobody NB7 blocks PKCβ phosphorylation, and phosphorylation prevents nanobody binding.

A. Extracted ion chromatograms for p110γ, p110γ-p101, and p110γ bound to NB7 are shown for the S594 or S595 phosphorylation site in p110γ. A bar graph showing the intensities of phosphorylated and non-phosphorylated p110γ peptide (593–607) for p110γ (black), p110γ with NB7 (red) and p110γp101 (purple) are shown to the right of the extracted ion chromatograms (n=3, right). In all experiments in panels A+B, PKCβ was present at 500 nM. Significance is indicated by ***(<0.0001%).

B. Extracted ion chromatograms for p110γ, p110γ-p101, and p110γ bound to NB7 are shown for the S582 phosphorylation site in p110γ. A bar graph showing the intensities of phosphorylated and non-phosphorylated p110γ peptide (579–592) p110γ (black), p110γ with NB7 (red) and p110γ-p101 (purple) are shown to the right of the extracted ion chromatograms (n=3, right). Significance is indicated by * (<0.01%), and ***(<0.0001%). The putative phosphorylation site is shown in red in the sequence above the bar graphs for both panel A+B.

C. Cartoon schematic of BLI analysis of the binding of immobilized His-NB7 to phosphorylated and non-phosphorylated p110γ.

D. Association curves for phosphorylated and non-phosphorylated p110γ (25nM) binding to His-NB7 are shown (n=3).

E. ATPase kinase activity assays comparing the activation/inhibition of phosphorylated and non-phosphorylated p110γ (1000 nM) with or without nanobody (3000 nM final) in the absence of PIP₂ membranes. Significance is indicated by * (<0.05%), and NS (>0.05%).