Iblot nitrocellulose membrane

Lysate was prepared from B16F10 cells using RIPA buffer containing 1X protease inhibitor cocktail (Millipore Sigma P8340) and boiled in 1X NuPage LDS sample buffer (Invitrogen NP0007) with 2.5% β-mercaptoethanol. Proteins were separated by electrophoresis in NuPage 4–12% Bis-tris gels run with 1X MOPS buffer (Invitrogen NP0323) and transferred to an iBlot nitrocellulose membrane (Invitrogen IB301002). The membranes were blocked with 5% (w/v) non-fat milk in Tris buffered saline (TBS) plus Tween-20 (TBST) for 1 h and stained with 5% (v/v) mouse serum overnight at 4°C with agitation. The membranes were washed TBST and incubated with 1:500 secondary antibody conjugated with horseradish peroxidase (GRP) in 5% (w/v) milk in TBST for 1 h at room temperature with agitation. The membranes were then washed again three times with TBST. Membranes probed with HRP-conjugated secondary antibody were developed a 3,3’,5,5’-teramethylbenzidine (TMB) substrate (Genscript L002V or Millipore Sigma T0565). Developed membranes were scanned and then processed with ImageJ.

EVs (2 × 10⁹ in 24 µL) were mixed with 8 µL of sample buffer (0.5 M dithiothreitol, 0.4 M sodium carbonate, 8% sodium dodecyl sulfate and 10% glycerol) and heated at 70 °C for 10 min. The mixture was loaded onto a NuPAGE Novex 4–12% Bis-Tris Protein Gel (Invitrogen, NP0335BOX) and separated at 120 V in NuPAGE MES SDS running buffer (Invitrogen, NP0002) for 2 h. Proteins on the gel were transferred to an iBlot nitrocellulose membrane (Invitrogen, IB23001) for 7 min using the iBlot system. Membranes were blocked with Odyssey blocking buffer (LI-COR, 927-60004) for 1 h under gentle shaking. Afterwards, the membrane was incubated overnight at 4 °C with primary antibody solution (1:1000 dilution for anti-TSG101 [Abcam, ab30871], anti-Calnexin [ThermoFisher, PA5-19169] and anti-Syntenin-1 [Origene, TA504796]; 1:200 dilution for anti-CD81 [SantaCruz, sc-9158] and 1:10,000 dilution for anti-β-actin [Sigma, A5441]). The membrane was rinsed with PBS supplemented with 0.1% Tween 20 (PBS-T) for 3 times over 15 min and incubated with the corresponding secondary antibody (925–68070, 926–68071, 926–32210, 926–32211; 1:15,000 dilution for all, LI-COR) for 1 h. Membranes were rinsed with PBS-T for 3 times over 15 min, one time with PBS and visualized on the Odyssey infrared imaging system (LI-COR, US).

Lysate was prepared from B16F10 cells using RIPA buffer containing 1× protease inhibitor cocktail (Millipore Sigma P8340) and boiled in 1× NuPage LDS sample buffer (Invitrogen NP0007) with 2.5% β-mercaptoethanol. Proteins were separated by electrophoresis in NuPage 4–12% Bis-Tris gels run with 1× MOPS buffer (Invitrogen NP0323) and transferred to an iBlot nitrocellulose membrane (Invitrogen IB301002). The membranes were blocked with 5% (wt/vol) non-fat milk in Tris-buffered saline (TBS) plus Tween-20 (TBST) for 1 hr and stained with 5% (vol/vol) mouse serum overnight at 4°C with agitation. The membranes were washed with TBST and incubated with 1:500 secondary antibody conjugated with horseradish peroxidase (HRP) in 5% (wt/vol) milk in TBST for 1 hr at room temperature with agitation. The membranes were then washed again three times with TBST. Membranes probed with HRP-conjugated secondary antibody were developed a 3,3’,5,5’-teramethylbenzidine substrate (Millipore Sigma T0565). Developed membranes were scanned and then processed with ImageJ.

Proteins from cells or exosomes were extracted with RIPA buffer (R0278, Sigma). The protein concentration was determined using a Bradford microassay (Bio-Rad Laboratories, Hercules, CA), and proteins were separated by SDS-PAGE and ten transferred to iBlot nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). Membranes were blotted with primary antibodies against CD63 (Santa Cruz Biotechnology), CD81 (Santa Cruz Biotechnology), TSG101 (ProteinTech, Chicago, USA), ANGPTL3, GAPDH (Abcam, Cambridge, UK), PI3K, AKT, phosphorylated PI3K (p-PI3K), and AKT (p-AKT) (Cell Signal Technology). After washing with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (Amersham Pharmacia). The immunoreactive bands were detected using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) and imaged with a ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad).