Anti nrp1

Recombinant proteins and cell lysate proteins were detected using Western blotting. Before harvesting, MH7A cells and FLS were washed with PBS. The cells were homogenized in 2% sodium dodecyl sulfate sample buffer using BioMasher II (Nippi Inc., Tokyo, Japan). The samples were then centrifuged for 5 min at 15,000× g and 20–25 °C. Proteins were processed using a SuperSep Ace 10% precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred onto a polyvinylidene fluoride membrane. The membranes were probed with anti-β-actin (mouse monoclonal, clone AC-15; Sigma-Aldrich), anti-NRP1 (rabbit monoclonal, clone EPR3113; Abcam), and anti-NRP2 (rabbit polyclonal; Sigma-Aldrich) antibodies. Rabbit sera against anti-nucleocapsid (N) protein were prepared by immunizing rabbits with the N-terminal domain of SARS-CoV-2 N protein expressed and purified in E. coli. Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were then added. Horseradish peroxidase activity was detected using ECL prime reagents (Cytiva, Tokyo, Japan), followed by imaging using an Image Quant LAS 500 (Cytiva) system.

Cells were lysed with ice-cold radioimmunoprecipitation assay buffer for 30 min (RIPA, Beyotime Biotechnology, Shanghai, China). The protein concentration was measured by BCA assay (Beyotime Biotechnology, Shanghai, China). Protein lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Millipore, Billerica, MA, USA), transferred onto nitrocellulose membranes, and immunoblotted with primary antibodies, followed by the matched secondary antibodies. Primary antibodies used in this study were anti-α-SMA (1:1000; Bioword Technology, MN, USA), anti-Vimentin (1:1000; Bioword Technology, MN, USA), anti-β-catenin (1:1000; Bioword Technology, MN, USA), anti-PI3K (1:1000; Bioword Technology, MN, USA), anti-STAT3 (1:1000; Bioword Technology, MN, USA), anti-VEGF-165 (1:1000; Biosson, Beijing, China), anti-Akt (1:1000; Cell Signaling Technology, Shanghai, China), anti-p-Akt (1:1000; Cell Signaling Technology), anti-mTOR (1:1000; Cell Signaling Technology), anti-p-mTOR (1:1000; Cell Signaling Technology), anti-CXCR4 (1:1000; Bioword Technology, MN, USA), anti-N-cadherin (1:1000; Bioword Technology, MN, USA), anti-NRP1 (1:1000; Abcam, Cambridge, MA, USA), and anti-GAPDH (1:5000; Proteintech Group Inc., Chicago, IL, USA).

Proteins were collected from cultured cells with SDS lysis buffer (2.2% SDS, 50 mm Tris/HCl pH 6.8, 1 mm PMSF 5.5% glycerol), and the cells were washed twice with 1X PBS before isolation. Proteins from the exosomes were resuspended in SDS lysis buffer after washing in 1X PBS. All protein collection steps were performed at 4 °C. Then, the lysates were heated at 95 °C for 5 min, quantified by a NanoDrop 2000, loaded with β‐Blue (20% β‐mercaptoethanol and 0.08% bromophenol blue) and stored at −80 °C. Thirty to fifty micrograms of protein in each well was used in all experiments (the same quantity of sample in the same experiment was used for each cell line), which was separated via SDS/PAGE and transferred onto polyvinylidene fluoride membranes (PVDF, Roche, Basel, Switzerland). Then, the membranes were incubated with antibodies at 4 °C overnight. The next day, the membranes were incubated with secondary antibodies for 1 h at room temperature. Antibodies, including anti‐CD63 (1:200; Santa Cruz, Dallas, CA, USA; sc‐5275), anti‐TSG101 (1:200; Santa Cruz, sc‐7964), anti‐CPT1A (1:1000; Abcam, Cambridge, UK; ab234111), anti‐NRP‐1 (1:1000; Abcam), anti‐Alix (Abcam) and anti‐CD9 (Abcam), were used to analyse the different proteins, and a β‐actin antibody (1:500; Santa Cruz, sc‐47778) was utilized for normalization.

To detect FABP3 and NRP-1, MBs, GCPs and differentiated GCPs cells were seeded on Lab-Tek Flask previously treated with Poly-D-lysine (Sigma-Aldrich, Saint Louis, MO, USA) and allowed to adhere for 16 h. Cells were fixed with 4% paraformaldehyde for 15 min at RT, permeabilized with 1xPBS, 0.1% triton X-100 for 2 min on ice and incubated in 1xPBS 5% bovine serum albumin (BSA). Cells were incubated overnight with anti-NRP-1 (monoclonal 1:300; Abcam, Cambridge, UK) and anti-FABP3 (H-FABP; polyclonal 1:100; Abcam, Cambridge, UK), and subsequently with the secondary antibody Alexa Fluor 488 (1:1000; Invitrogen) and DAPI (1:1000; Thermo Fisher, Waltham, MA, USA). Images were acquired with an Eclipse 80i Fluorescence Microscope (Nikon, Tokyo, Japan), equipped with the Imaging Software NIS-Elements BR Version 3.2.