Pierce protein concentrator pes 3 k mwco

Whole cell lysates were created using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Thermo Scientific). Conditioned media was collected after 48-h of culture in the presence or absence of doxycycline. The conditioned media was cleared of cellular debris by centrifugation and concentrated using the Pierce protein concentrator PES 3 K MWCO (ThermoFisher). Samples were quantified and equal amounts of protein lysate or conditioned media were separated on 4–12% Bis–Tris gradient gels (Thermo Scientific). The proteins were transferred to polyvinylidene fluoride membranes, blocked with 5% bovine serum albumin, and probed with primary antibodies against the C-terminus of WT1 (sc-192; Santa Cruz), FGFR4 (#8562; Cell Signaling), GAPDH (#5174; Cell Signaling), IGF2 (ab9574; Abcam), CD200 (AF2724; R&D Systems), or B7-H3 (#14058; Cell Signaling). The membranes were incubated with HRP conjugated secondary antibodies (anti-rabbit and anti-goat) and subsequently visualized with ECL reagent (Thermo Scientific). The blots were imaged using the ChemiDoc MP system (Bio-Rad). All blots were performed in biological triplicate experiments and representative images are shown.

Exosomes were purified from the conditioned medium using ultracentrifugation as described by Breglio et al., 2020 (link). Briefly, conditioned medium was first centrifuged at 300× g for 10 min at 4°C, then at 10,000× g for 30 min at 4°C. Finally, exosomes were pelleted by ultracentrifuging at 100,000× g for 70 min at 4°C. The exosome-depleted supernatant was further concentrated with a Pierce Protein Concentrator PES (3 K MWCO, Cat#: 88525, ThermoFisher) at 4000× g and 4°C. Both the concentrated non-exosomal supernatant and the purified exosomes were resuspended in RIPA Lysis and Extraction Buffer supplemented with cOmplete Mini EDTA-Free Protease Inhibitor Cocktail (Roche) and vortexed for 1 min. Protein concentration was analyzed with a BCA protein assay kit. The remaining sample was denatured at 95°C for 5 min in 4×Laemmli buffer and subjected to SDS-PAGE using 4–15% Mini-PROTEAN TGX Precast Protein Gels followed by protein transfer to a 0.45 μm pore (polyvinylidene difluoride) PVDF membrane (Cat#: 88585, ThermoFisher). Membranes were blocked in 3% BSA in TBS with 0.1% Tween-20 (TBS-T). The primary Abs used were VEGFA (1:400) and PDGFRβ (1:5000). HRP-linked secondary Ab: Goat Anti-rabbit IgG (1:10,000). Protein bands were visualized by chemiluminescence using a SuperSignal West Femto Duration Substrate and Q-View Imager System (Quansys Bioscience, Logan, UT, USA).