Tnrc6a

Cell lysate was prepared with RIPA buffer as mentioned above, incubated with antibody against TRIM65 (Abcam), TNRC6A (Abcam) or IgG (Santa Cruz Biotech., Santa Cruz, CA, USA) for 2 h, and then with protein A/G Plus agarose beads (Santa Cruz Biothech.) for 1 h. Immunoprecipitates were washed three times with RIPA buffer, fractionated by SDS-PAGE, and detected by western blotting with primary antibodies against TRIM65, TNRC6A, and ubiquitin (Abcam).

After washing twice with ice-cold phosphate-buffered saline (PBS), the cells were incubated at 4 °C with RIPA buffer supplemented with a proteinase inhibitor cocktail (JRDUN biotech, Shanghai, China) for 30 min. Subsequently, the samples were centrifuged at 12,000 r.p.m. for 15 min at 4 °C, and the supernatant was collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein on the gels were electrophoretically transferred to a nitrocellulose membrane (Millipore, Bredford, MA, USA), and probed with primary antibodies against TRIM65 (Abcam, Cambridge, MA, USA; 1:1000 dilution), TNRC6A (Abcam; 1:1000 dilution), ATG7 (Abcam; 1:200 dilution), LC3 (Abcam; 1:2000 dilution) or cleaved caspase3 (Abcam; 1:1000 dilution). After incubation with an HRP-conjugated secondary antibody (Beyotime, Shanghai, China; 1:1000 dilution), signals were detected using an enhanced chemiluminescence (ECL) detection kit (Millipore). The membrane was probed with a primary antibody against GAPDH (Cell Signaling Technology, Danvers, MA, USA; 1:2000 dilution) for equal loading control.