Peroxidase linked anti rabbit igg or anti mouse igg secondary antibodies

ALCAR ability to induce apoptosis in PCa and BPH cells was confirmed by western blotting. Following 24 h of treatment with ALCAR (1 or 10 mM), cells were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Proteins (30 μg) were separated on the NupageNovex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a PVDF membrane Amersham Hybond (GE Healthcare Biosciences). Membranes were incubated overnight at 4 °C with Cleaved Caspase-3 (Asp175) (Cell Signalling Technology) and with peroxidase-linked anti-rabbit IgG or anti-mouse IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Protein expressions were normalized to beta-Actin (Abcam). Band intensity (revealed as optical density-OD) were detected by ImageJ software.

EVs were also characterized by Western blotting for the expression of specific markers. EVs were lysed in radioimmunoprecipitation assay buffer (RIPA buffer), and supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Proteins (30 μg) were separated on the Nupage Novex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a Polyvinylidene fluoride (PVDF) membrane Amersham Hybond (GE Healthcare Biosciences, Piscataway, NJ, USA). Membranes were incubated overnight at 4 °C with primary antibodies anti-CD81, anti-CD63, anti-αHSP70 (ExoAb Antibody Kit, System Biosciences, Palo Alto, CA, USA), anti-TSG-101 (Thermo Fisher Scientific), anti-MyoD1 (Abcam), anti-MHC (R&D) followed by peroxidase-linked anti-rabbit IgG or anti-mouse IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).