Ab175200

Western blot was conducted according to previously described.¹⁷ RIPA buffer (Beyotime, China) containing protease inhibitor (Thermo Fisher Scientific, USA) was used to isolate total proteins. Then, the concentration of proteins was evaluated by BCA Protein Assay kit (Thermo Fisher Scientific). Sodium dodecylsulphonate (SDS) polyacrylamide gel electrophoresis was used to separate proteins, followed by electroblotting (Millipore, USA) which can transfer proteins onto a polyvinylidene fluoride (PVDF) membrane. Next, PVDF membranes were incubated with primary antibodies for a night at 4°C. The following antibodies were employed to examine target proteins: anti‐OTUB1 monoclonal antibody (1:500; ab175200, Abcam), anti‐EYA1 polyclonal antibody (1:500, ab194448, Abcam) and anti‐ubiquitin monoclonal antibody (1:500, sc8017, Santa Cruz). After incubation with secondary antibody (Proteintech, China) for 1 h at 37°C, protein band intensity was evaluated through Quantity One software (Bio‐Rad, USA).

The following antibodies were used in WB analysis: anti-ubiquitin (ab134953; Abcam, Cambridge, UK), SLC7A11 (D2M7A; Cell Signaling, Danvers, MA, USA), anti-OTUB1 (ab175200; Abcam, Cambridge, UK), anti-CD44 (ab189524; Abcam), anti-SLC7A11 (ab175186; Abcam), anti-CD44 (ab119348; Abcam), rabbit monoclonal [EPR5702] to CD63 (ab134045; Abcam), rabbit monoclonal [EPR4244] to CD81 (ab109201; Abcam), GAPDH Monoclonal antibody (60004-1-Ig; Proteintech), Beta Tubulin Polyclonal antibody (10094-1-AP; Proteintech) and goat anti-rabbit IgG H&L (HRP) (ab6721; Abcam).