Phostop easy pack phosphatase inhibitor

After treatment, ARPE-19 cells were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride and PhoSTOP EASY pack phosphatase inhibitor (Roche, Mannheim, Germany) on ice for 30 min. The lysate were clarified by centrifugation at 12000 rpm for 5 min. Total protein concentration was quantified by a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA). 50 μg protein was loaded and separated on 6% and 10% SDS-PAGE gels and transferred onto nitrocellulose membrane (Bio-rad, CA, USA). To avoid non-specific binding, the membranes were blocked using 5% bovine serum albumin (BSA, Sigma Aldrich, MO, USA) in PBS for 45 min at room temperature. The membranes were then incubated with primary antibodies diluted in 2% BSA in PBS with 0.1% Tween-20 (PBS-T) at 4°C overnight. After rinsing with PBS-T for three times, the membranes were incubated with IRDye® 680LT Goat anti-Rabbit or IRDye® 800CW Goat anti-Mouse secondary antibodies (Li Cor Biosciences, NE, USA) at room temperature for 1 h. After washed with PBS-T for three times, the bound antibody was detected by Odyssey infrared imaging system (Li Cor Biosciences, NE, USA). The band intensities are analyzed to with the Odyssey software and normalized to β-actin or GAPDH.

Cells/exosomes were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulphonyl fluoride and PhoSTOP EASY pack phosphatase inhibitor (Roche, Mannheim, Germany) on ice for 30 m, 48 hours after treatment. The lysates were clarified by centrifugation at 8317 g for 5 m at 4°C. Total protein concentration was quantified by a bicinchoninic acid assay kit (Thermo Scientifics), and 40 μg protein was loaded and separated on SDS‐PAGE gels and transferred onto nitrocellulose membrane (Bio‐rad). The membranes were blocked using 5% BSA (Sigma‐Aldrich) in PBS for 45 m at room temperature to prevent non‐specific binding. The membranes were then incubated with primary antibodies diluted in 2% BSA in PBS with 0.1% Tween‐20 (PBS‐T) at 4°C overnight. After rinsing with PBS‐T for three times, the membranes were incubated with IRDye^® 680LT Goat anti‐rabbit or IRDye^® 800CW Goat anti‐mouse secondary antibodies (Li‐Cor Biosciences) at room temperature for 1 hour. After three washes with PBS‐T, the bound antibody was detected using an Odyssey infrared imaging system (Li‐Cor Biosciences). The band intensities were analysed using Odyssey software and normalized to β‐actin or GAPDH.