Halt protease inhibitor single use cocktail

WJMSCs and sEVs were lysed with RIPA buffer containing a Halt protease Inhibitor single‐use cocktail (Thermo Scientific). Membrane‐bound PD‐L1 were extracted using Mem‐PER™ plus membrane protein extraction kit following the manufacturer's instruction (Thermoscientific, USA). Protein lysates were separated by a Mini‐protean TGX precast gel (BIO‐RAD, USA) and transferred onto a PVDF membrane (BIO‐RAD, USA). Western blots were performed according to the standard techniques. The following antibodies were used at a dilution of 1:1000 in 5% nonfat milk unless otherwise stated: goat anti‐human PD‐L1 and PDL2 (R&D), Rabbit anti‐human CD90, CD105 (Thermo Scientific), Rabbit anti‐human CD9, CD81, HSP70, and CD63 (System Biosciences) and mouse anti‐human β‐actin (1:10,000, Sigma). Secondary antibodies include donkey anti‐goat‐HRP (R&D), goat‐anti‐rabbit‐HRP (Cell Signalling) and goat anti‐mouse antibody conjugated with HRP (Sigma). Signals were developed by using Pierce ECL Western Blotting Substrate (Thermo Scientific).

The recombinant NP antigens were generated and purified as previously described [16 (link)]. Briefly, human embryonic kidney 293T (HEK293T) cells were transfected with the pCAGGS plasmids encoding NPs, using TransIT-LT1 Transfection Reagent (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer’s protocol. After 48-h incubation, the cells were harvested by pipetting, washed with phosphate-buffered saline (PBS), and treated with lysis buffer (10 mM Tris-HC_l, pH 7.8, 0.15 M NaCl, 1.0 mM EDTA, and 0.25% NP-40) in the presence of halt protease inhibitor single use cocktail (Thermo Scientific, Waltham, MA, USA), and recombinant NPs were purified from the cell lysate through ultracentrifugation with 20%–50% (w/v) discontinuous CsC_l gradients. The NP fractions were pooled, and purified NP was collected by ultracentrifugation. The concentration of NP was determined using the bicinchoninic acid assay (Pierce Micro BCA Assay Kit-Perce) (Thermo Scientific, Rockford, IL, USA) according to the company instructions and used for enzyme-linked immunosorbent assay (ELISA) and immunization of mice.

Total cellular proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) containing halt protease inhibitor single-use cocktail (Thermo). The extracted total proteins were denatured by adding 5× sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (Thermo), followed by boiling for 5 min at 100°C. Approximately 15 μg proteins was applied for SDS-PAGE (Wang et al., 2017 (link)). The primary antibodies used in this study included antibodies against β-actin, p-JNK, JNK, SQSTM1/P62, LC3B (Cell Signaling Technology, Beverly, MA, USA), CVA16 (Millipore, MA, USA) and EV71 VP1 (Abnova, Taipei, China). The goat anti-rabbit and anti-mouse HRP-labeled antibodies were obtained from Cell Signaling Technology.

SDS-PAGE and WB were performed according to standard protocols, as described [58 (link)]. Briefly, cells were lysed in lysis buffer containing 15 mM Tris/HCl pH 7.5, 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton 100, Halt Protease Inhibitor Single-Use cocktail (100×, Thermo Scientific). Whole cells lysates (50 μg per sample) from transfected cell lines were separated using 4–12% Novex Bis-Tris SDS-acrylamide gels (Invitrogen), electro-transferred on nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and immunoblotted with the following antibodies: PSMβ5 (D1H6B) Rabbit mAb (Cell Signaling), Caspase-3 (8G10) Rabbit mAb (Cell Signaling), Caspase-7 (C7) Mouse mAb (Human Specific), γ-Tubulin antibody (C-20) goat polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH (D16H11) XP® Rabbit mAb. Membranes were washed 3 times in PBS-Tween, and then incubated with a secondary antibody conjugated with horseradish peroxidase in 5% milk for 2 h at room temperature. Chemiluminescence was detected using Pierce ECL Western Blotting Substrate (Pierce Biotechnology, Waltham, MA, USA).

SDS-PAGE and western blot (WB) were performed according to standard protocols. Briefly, after respective transfection and LIPUS treatments, hMCSs cells were lysed in lysis buffer containing 15 mM Tris/HCl pH 7.5, 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton X100, and a Halt Protease Inhibitor Single-Use cocktail (100×, ThermoFisher Scientific, Rodano, Italy). Whole lysate (15 µg per lane) was separated using 4–12% NovexBis-Tris SDS-acrylamide gels (Invitrogen, Life Technologies), electro-transferred on nitrocellulose membranes (Bio-Rad Laboratories Srl, Segrate, Milan, Italy), and immunoblotted with the appropriate antibodies. Antibodies against the following proteins were used: HIF-1α (anti-rabbit HIF-1α, Merck Millipore SpA, Vimodrone, Milan, Italy), RhoA (NB100-91273, NovusBiological, Milan, Italy), VHL-2 (VHL (FL-181): sc-5575, Santa Cruz Biotechnology, INC., Heidelberg, Germany), and α-Actin (monoclonal anti-α-actin (1A), sc32251, Santa Cruz Biotechnology, Inc.). The secondary antibodies used for immunoblotting were obtained from ThermoFisher Scientific (ThermoFisher Scientific, Rodano, Italy) and signals were detected using a CCD high-resolution and high-sensitivity detection technology (ChemiDoc™ XRS+ System, Bio-Rad Laboratories Srl, Segrate, Milan, Italy).