Fig. 4
From: Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery
Western blots of the BirA assay showing increased delivery of LFN-cargo constructs with PAwt-sANTXR-Ac2 on Flp-In 293-EpCAM-BirA cells. Cytosolically delivered cargo proteins are biotinylated by a cytoplasmically encoded BirA and stained with Streptavidin IRDye 680LT. Total cellular uptake measured via HA-tag on the LFN-cargo. a, b Increasing concentrations of respective PA constructs incubated with a 5-fold excess of LFN-NI1C. Boxes indicate the bands of interest. c, d Quantification of western blot bands from a and b. Black bars, PAwt-sANTXR-Ac2; red bars, PAm-sANTXR-Ac2; green bars, PAm-Ac2. The dotted line represents background signal (i.e., cells only), and the dashed line shows the signal of cargo at 20 nM PAwt-sANTXR-Ac2. e, f Cytosolic localization (e) and total cellular uptake (f) of three different cargo DARPins delivered with 20 nM (lanes 1–3) and 100 nM (lanes 4–6) of PAwt-sANTXR-Ac2 or 100 nM (lanes 7–9) for PAm-Ac2; lanes 10 and 11 represent cells incubated with 100 nM LFN-cargo without PA. "dest." refers to rationally destabilized versions of NI2C and NI3C DARPins [10]. Boxes indicate the bands of interest