Sox11 antibody

The proteins were extracted from BC cells by RIPA buffer (Beyotime, Beijing, China) and quantified with a Protein Quantification kit (Millipore, Billerica, MA, USA). The cell extracts were subjected to Western blot to determine the protein expression of SOX11 and Ago2. GAPDH was used as the protein loading control. Equal amounts of protein extracts were loaded on to SDS-PAGE gel and transferred to the PCDF membrane. After transfer, the membrane was blocked with 5% skim milk, followed by incubation with primary antibodies, SOX11 antibody (1:1000; Abcam, Shanghai, China), Ago2 antibody (1:1000; Abcam, Shanghai, China) or GAPDH antibody (1:1000; Santa Cruz, Santa Cruz, CA, USA). Subsequently, the membrane was washed and incubated with goat anti-rabbit IgG H&L (HRP) secondary antibody (1:2000; Abcam, Shanghai, China). ECL Reagent (Cell Signaling Technology, Danvers, MA, USA) was used for visualization and detection.

The transfected AT2 cells were solubilized in RIPA cell lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with a protease inhibitor cocktail and PMSF on ice for 15 min. Lysates were centrifuged at 14,000 × g for 15 min at 4°C. To assess the interaction between SOX11 and FAK, the supernatant was incubated overnight at 4°C with SOX11 antibody (Abcam; cat. no. ab170916). Subsequently, 40 µl protein A cross-linked to agarose beads (EMD Millipore; cat. no. IP05) were added, and the mixture was incubated for 1 h at 4°C with constant rotation. The beads were washed 6 times with ice-cold lysis buffer prior to the addition of SDS-PAGE sample buffer to each sample. The bound proteins were dissociated from the beads by heating at 92°C for 3 min before they were resolved on 10% SDS-gels using SDS-PAGE as described above. Western blot analysis was used to evaluate the expression of SOX11 and FAK as described above.