Mouse anti human β actin

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).

Fibroblast cells (healthy or asthmatic) were collected and washed with PBS, after which the proteins were extracted using the laemmli or RIPA lysis buffer (Sigma-Aldrich, Germany). All protein extracts were quantified using Bradford Protein Assay Kit, according to the manufacturer’s instructions (Bio-Rad, United States). 10 μg (for fibroblasts) of protein was separated on SDS-PAGE and transferred to a nitrocellulose membrane. Expressions of β-catenin and β-actin were assessed using rabbit anti-human CTNNB1 (Cell Signaling, United States) and mouse anti-human β-actin (A5441, Sigma, Germany), respectively. Anti-rabbit and anti-mouse IgG HRP-linked antibodies (Cell Signaling, United States) were used along with Clarity Western ECL Substrate (Bio-Rad, United States) for chemiluminescent detection of protein bands. Western blot analysis of cell fractions from asthmatic bronchial fibroblast using Cell Fractionation Antibody Sampler Kit #11843 showing cytoplasmic (C.F), organellular/membrane (M.F), and nuclear/cytoskeletal localization (N.F.). Whole-cell lysates (WCL) represent total protein. The fractionation was done under two conditions, the cultivation with high glucose medium and low glucose medium.

Protein samples were separated on an SDS-PAGE, followed by transferring to a nitrocellulose membrane as described previously [30 (link)]. Protein bands were detected using the following primary antibodies: mouse anti-human β-actin (1:5000 dilution, Sigma-Aldrich); rabbit anti-human phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000 dilution) and p44/42 MAPK (Erk1/2) (1:1000 dilution); rabbit anti-human phospho-Akt (Ser473) (1:2000 dilution) and Akt (1:1000 dilution) (Cell Signaling Technology, MA, USA). Blotted protein bands were detected with respective horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence (ECL) western blot analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Western blotting was carried out as previously described [25 (link)]with some modifications. Briefly, cells were lysed in lysis buffer (50mM Tris.HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 0.001% bromophenol blue, 100mM DTT, 10X protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma)). Samples were heated to 95°C for 10 minutes and stored at -80°C. Lysates were subjected to SDS/PAGE on 15% Tris-Tricine Criterion (Bio-Rad, Hercules, CA) minigels followed by transfer to 0.2-μm-pore polyvinylidene difluoride membrane (Immun-Blot PDVF membrane; Bio-Rad). Membranes were probed with antibodies to mouse anti-human LC3 (2G6, nanoTools Antikörpertechnik GmbH, Teningen Germany) and mouse anti-human β–actin (Sigma). Following exposure to HRP-conjugated goat anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA, USA), proteins were visualized by enhanced chemiluminescence using Supersignal West Pico chemiluminescent substrate (ThermoScientific, Rockford, IL, USA).

Platelets samples were lysed (NaCl 0.15 M, Tris 10 mM pH 8, EDTA 0.1 mM, Triton X-100 1%, and protease inhibitor cocktail (ROCHE)) and protein quantification was performed using BCA kit (Thermo Scientific). Protein extracts (10 µg) from platelets from 4 patients and 4 healthy volunteers were prepared with sample buffer containing β-mercaptoethanol, separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into nitrocellulose membrane. The membranes were blocked with Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBS-T) plus 5% milk for 1 hour and incubated with primary antibodies mouse anti-iNOS (BD610333) and mouse anti-human β-actin (Sigma A1978).

Freshly isolated platelets from HIV-infected subjects or healthy volunteers were kept unstimulated or stimulated with 0.4 U/mL of thrombim for 1 h. Platelets were them lysed (0.15MNaCl, 10 mM Tris pH 8.0, 0.1 mM EDTA, 10% Glicerol and 0.5% triton X-100) in the presence of protease inhibitors (Roche, Indianapolis, IN). Platelet proteins
(17.5 μg) were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into nitrocellulose membrane. The membrane was blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBS-T) and 5% low-fat dried milk for 1 h before incubation overnight with mouse anti-human PF4/CXCL4 (R&D Systems) (1:1000) or for 1 h with mouse anti-human β-actin (Sigma Aldrich) (1:10,000) primary antibodies. After washing five times in TBS-T, the membrane was revealed using peroxidase-conjugated secondary antibodies against mouse immunoglobulin (Vector) (1:10,000, 1 h).

Western blot analysis was performed as previously described [6 (link)] with the following antibodies: monoclonal anti-Gli1 (Novus Biologicals, Littleton, CO), monoclonal anti-total STAT3 (BD Biosciences Pharmingen, San Diego, CA), monoclonal anti-serine STAT3 (BD) and mouse anti-human β-actin (Sigma-Aldrich). Densitometry analysis was performed using an Epson Expression 1680 scanner (Epson America, Inc.). Densitometry values were normalized to β-actin.