Immobilon western chemiluminescent hrp substrate detection system

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, 2 mM l-glutamine, lipofectamine 2000, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture dishes, 6-well, and 12-well plates were purchased from Greiner Bio-One (Frickenhausen, Germany). Polyvinyldifluoride (PVDF) membrane and an Immobilon Western Chemiluminescent HRP Substrate detection system were purchased from Millipore (Billerica, MA, USA). All of the primary antibodies specific for IL-6 (sc-28343), CXCR2 (sc-32780), c-Raf (sc-7267), MEK (sc-6250), ERK (sc-514302), JNK (sc-7345), p38 (sc-81621), c-Jun (sc-166540), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal rabbit antibodies specific for phosphorylated forms of c-Raf (#9427), MEK (#3958), ERK (#4376), JNK (#9255), p38 (#9216), and c-Jun (#2361) were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). All inhibitors against signal pathway components were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human CXCL1 was obtained from PeproTech (Rocky Hill, NJ, USA). All small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Plasma-derived exosomes were lysed using ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF and protease inhibitor (SigmaFast, Sigma-Aldrich, St. Louis, MO, USA). Proteins (20 μg) of each sample were diluted 1:1 with Laemmli buffer supplement with dithiothreitol (50 mM) and denatured by boiling for 5 min. Proteins were separated on 12% SDS-PAGE gels and transferred to a PVDF membrane (Immobilon^®-P, Millipore, Billerica, MA, USA). Membranes were incubated with specific primary antibodies: anti-CD81 (1:1000), anti-CD9 (1:1000), anti-Hsp70 (1:1000), anti-caveolin-1 (1:1000), anti-caveolin-3 (1:1000), and anti-hnRNPA2B1 (1:750) with milk at 3% overnight at 4 °C. Posteriorly, HRP-conjugated secondary antibody was used (1:15,000, Jackson ImmunoResearch Lab., West Grove, PA, USA) for 1 h at room temperature with constant agitation. The immunoblotted proteins were detected with Immobilon™ Western Chemiluminescent HRP Substrate detection system (Millipore, Billerica, MA, USA). All images were analyzed using ImageJ (NIH, Bethesda, MA, USA) and reported as arbitrary units.

Cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with anti-protease and anti-phosphatase cocktail mixes and protein concentration was determined with the Bradford reagent (all from Sigma-Aldrich). Fifty to eighty micrograms of each sample were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond ECL, Amersham, Milan, Italy). Membranes were blocked with 3% non-fat milk for 30 min and processed for immunodetection using specific horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) and the Immobilon Western Chemiluminescent HRP substrate detection system (Millipore). Densitometric analysis of band intensity was carried out with the Image J Software developed by NIH and freely available on the web.