Ab82411

Total protein concentration in the plasma samples was determined using Pierce BCA Protein Assay (Thermo Fisher, Waltham, MA). General protein separation was carried out by electrophoresis on 4–20% gradient SDS-PAGE gels at 110 V for 1.25 h and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Transfer membranes were then incubated in blocking buffer (1% BSA in TBST) at 4 °C under gentle agitation overnight. Blocked membranes were probed with primary recombinant monoclonal antibodies for trypsin (ab200997, 1:1000, Abcam, Cambridge, MA), pancreatic lipase (ab124915,1:30000, Abcam), and Transferrin (ab82411, 1:10,000, Abcam) before incubated in the corresponding secondary antibody (ab205718, 1/5000, Abcam). Membranes were incubated with Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and then imaged via photo-sensitive autoradiography film (Genesee Scientific, San Diego, CA). Western blot images were digitally analyzed (gel analysis tool, Image J; NIH). The band density values for each of the western blots for trypsin and lipase were normalized to their respective transferrin values.

For co-culture experiments, AML-12 cells at a density of 2 × 10⁵ cells per ml were cultured in a transwell system with a 0.4-μm porous membrane. For EVs uptake experiments, Raw264.7-derived EVs were labelled with the PKH67 Green Fluorescent Cell Linker Kit (Sigma Aldrich, St. Louis, MO, USA): EVs diluted in PBS were added to 1 ml Diluent C. In parallel, 4 μl PKH67 dye was added to 1 ml Diluent C and incubated with the EVs solution for 4 min. Then 2 ml 0.5% BSA/PBS was added and the labeled EVs were washed at 100,000 g for 1 h, the EVs pellet was diluted in 100 μl PBS and used for uptake experiments. The transferrin neutralized antibody (1:100, ab82411, Abcam, Cambridge, UK) was used to block the transferrin in hepatocytes.

Urine proteins were prepared as described in Materials and Methods; 20 µg of each sample were separated by 10% SDS-PAGE and transferred to PVDF membranes (Whatman, Maidstone, UK) in transfer buffer (10% methanol, 25 mM Tris base, 192 mM glycine, PH 8.0). Membranes were incubated overnight at 4 °C with primary antibody against alpha-1-antiproteinase (Species reactivity: rat; dilution 1:1000; ab106582, Abcam, Cambridge, UK) or transferrin (Species reactivity: rat; dilution 1:10000; ab82411, Abcam, Cambridge, UK). The membranes were then washed and incubated with peroxidase-conjugated anti-chicken and anti-rabbit IgG (1:10000; zSgb-Bio, Beijing, China) at room temperature for 2 h and proteins were visualized using enhanced chemiluminescence (ECL) reagents. Intensity of each protein band was quantified using Image J analysis software (National Institutes of Health, Bethesda, Maryland, USA).

Biotinylated protein levels in mouse tissues were assessed by immunoblotting. Tissue lysates were denatured at 95 °C for 5 minutes in 4X Laemmli sample buffer with 10% β-mercaptoethanol. Samples were loaded on Criterion™ TGX™ any KD gels and run in Novex® Tris-Glycine SDS running buffer. Samples were then transferred to Trans-Blot^® Turbo™ Midi PVDF using the Bio-Rad rapid semi-dry system. After Ponceau S staining, membranes were blocked in 2% non-fat milk in Tris-buffered saline with Tween-20^® for 1 hour at room temperature. Membranes were incubated overnight at 4 °C with the following primary antibodies at 1:5,000 dilution in blocking solution: Rabbit polyclonal anti-biotin (ab1227, Abcam), rabbit polyclonal anti-transferrin (ab82411, Abcam), rabbit polyclonal anti-BSA (A11133, ThermoFisher Scientific), rabbit polyclonal anti-GAPDH (ab9485, Abcam), rabbit polyclonal anti-beta tubulin (ab6046, Abcam). Goat anti-rabbit HRP secondary antibody or Pierce™ high sensitivity streptavidin-HRP antibody (21130, ThermoFisher Scientific) was added at 1:10,000 dilution in blocking solution and membranes were incubated for 1 hour at room temperature. Signal was visualized using SuperSignal™ West Dura Extended Duration Substrate or SuperSignal™ West Femto Maximum Sensitivity Substrate on HyBlot CL^® film.