Amersham imager 600 series imagers

Total exosomic and cellular protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Beijing, China). The protein concentrations of cell and exosome lysates were determined using BCA protein assay kit (Beyotime Institute of Biotechnology, Beijing, China). For Western blot analyses, the cellular and exosomic proteins (10 µg/well) were separated by electrophoresis in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM TrisCl, pH 8.0, 150 mM NaCl, 0.5% Tween 20) at 4 °C overnight, rinsed three times (10 min/time) with TBS-T, followed by 3 h incubation at room temperature with a mouse anti-human CD63 antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) or mouse anti-human β-actin antibody (1:10,000; Proteintech Group, Chicago, IL, USA), followed by 1 h incubation with HRP-conjugated rabbit anti-mouse IgG (Zymed Lab, San Francisco, CA, USA). The bound antibody was detected using Amersham Imager 600 series imagers (GE Healthcare, Chicago, IL, USA).

Total cellular proteins were prepared from the cells under different culturing conditions. Each of the sample proteins (30 µg) was added into the well and separated by means of 10% polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked by 5% skimmed milk in TBS-T (10 mM TrisCl, pH 8.0, 150 mM NaCl, 0.5% Tween-20) at room temperature for 2 h, rinsed gently with TBS-T, followed by incubation with the first antibody in appropriate concentrations (IL-13Rα2: 1:800, p-MKK7: 1:500, JNK: 1:800, p-JNK: 1:500, c-Jun: 1:800, Bak: 1:500, Bcl-2: 1:500, β-actin: 1:10,000) at 4 ℃ overnight, followed by 1 h incubation with HRP-conjugated goat anti-rabbit or anti-mouse IgG. Each step in the reaction with the antibody was followed by 10 min rinsing with TBS-T (three times). The bound antibody was detected using Amersham Imager 600 series imagers (GE Healthcare, Chicago, IL, USA). Each membrane was washed three times with TBS-T, blocked by 5% skimmed milk in TBS-T for 2 h and reused at least twice to identify different kinds of proteins or preserved at –20 °C.