Radioimmunoprecipitation assay (ripa)

Retinal tissues and Müller cells were collected in 1.5 mL centrifuge tubes, and 1 mL of radioimmunoprecipitation assay (Sigma-Aldrich, St. Louis, MO, USA) buffer was added. Retinal tissues and Müller cells were incubated on ice for 1 hour. Next, 1 μL (2 μg) of the primary antibody was added to the cell lysate supernatant after centrifugation (1000 × g, 5 minutes, 4°C), and the supernatant was incubated at 4°C for 1 hour. Afterward, 20 mL resuspended protein A/GPLUS-agarose (Santa Cruz, Deerfield Beach, FL, USA) was added to the cell lysate supernatant and incubated overnight at 4°C with shaking. The supernatant was discarded after centrifugation (1000 × g, 5 minutes, 4°C), and the agarose was washed three times with PBS for 5 minutes each and then boiled with 1× loading buffer (40 μL) for 10 minutes at 100°C. Finally, protein expression was assessed by western blotting. Isotype IgG was used as a negative control. The antibodies used are listed in Additional Table 2.

The radioimmunoprecipitation assay (Sigma-Aldrich) lysate and phenylmethylsulfonyl fluoride (Sigma-Aldrich) were mixed to extract the total proteins from the SIL-treated ACC-M cells and the lung metastases of ACC nude mice. The bicinchoninic acid method was used to detect the total proteins. The proteins were then mixed with 2× sodium dodecyl sulfate loading buffer and heated until denaturation. Fifty micrograms of proteins was loaded into each lane for a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterward, the proteins were transferred onto one polyvinylidene difluoride membrane, followed by closure with 3% skimmed milk at room temperature overnight; the primary antibodies (anti-β-actin antibody (Abgent, San Diego, CA, USA): diluted with Tris-buffered saline and tween 20 (TBST), 1:2,000; anti-LC3 antibody (Abgent): diluted with TBST, 1:500) were then added and incubated at 4°C overnight. After washing with TBST three times (slightly pendulated for 10 minutes each time), an horse radish peroxidase-conjugated goat antimouse IgG (Abgent) (dilution with TBST, 1:2,000) was added and incubated at room temperature for 1 hour. After subsequently washing with TBST three times for 10 minutes, the ECL kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for coloration and the films were then developed in darkness.