Ecl chemiluminescence working solution

RAW264.7 macrophage cell samples from the different groups were collected, washed three times with PBS and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) containing the halt protease inhibitor cocktail (100× stock diluted to 2×) (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration of the lysates was measured using a BCA protein assay kit. 15 μg of lysates were separated by 12% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After three washes with PBS, the membranes were blocked with 5% fat-free milk in PBST for 2 h at RT, then incubated with primary antibodies against C/EBP δ (1:1000 dilution in PBST) (Abcam, Cambridge, MA) at 4 °C overnight. The membranes were washed thrice with PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody for 1 h at RT. The membranes were washed with PBST three times, and the bands were detected using ECL chemiluminescence working solution (Beyotime, Shanghai, China), following the manufacturer’s instructions.

The protein concentration of exosome-like vesicles, soluble worm antigens (SAg) and ESP from T. pisiformis cysticercus were measured using a BCA protein assay kit. Fifteen μg of total protein was denatured at 100 °C for 10 min and separated by 12% SDS-PAGE. The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) for 13 min and blocked with 5% non-fat milk in PBST for 2 h at room temperature. Two antibodies of anti-14-3-3 and anti-enolase (1:200; both from T. solium produced in rabbits and were prepared in our laboratory) [36 (link)] were separately added to the membrane and incubated at 4 °C overnight. The membranes were washed three times with PBST and incubated with HRP-goat-anti rabbit IgG (H + L) (1:1000; Beyotime, Shanghai, China). The bands were developed using an ECL chemiluminescence working solution (Beyotime) following the manufacturer’s instructions.