Figure 6.

NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in Supplementary Figure S3. IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm

NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in Supplementary Figure S3. IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm