Ab80425

Total proteins were acquired from the transfected cells with protein lysis buffer (KeyGen, Nanjing, China) and extracted by Total Protein Extraction Kit (PROTTOT-1KT, Sigma-Aldrich). The protein extractions were treated using an SDS Quick Match Gel Kit (P0670, 250 mL, Beyotime Biotechnology, Shanghai, China) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Vicmed, China). The PVDF membranes were subsequently blocked with 5% skimmed milk and incubated with anti-CD44, anti-SOX2, anti-Oct4, anti-Nanog, anti-ZO-1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-U2AF2, anti-HNRNPC, anti-FUS, anti-DBCRB, anti-DDX54 (ab76947, Abcam), anti-NUCKS1 (ab80425, Abcam), anti-mTOR, anti-SREBP-1c, anti-Cyclin D1, or anti-GAPDH (KC Bio, China) antibodies, respectively, at 4°C overnight. Afterward, their corresponding secondary antibodies were incubated for another hour at room temperature. GAPDH acted as the internal reference.

Protein was extracted by RIPA lysis buffer (Beyotime). Protein samples were separated by separating gel, and then shifted onto a PVDF membrane. The membrane was mixed with primary antibodies at 4°C. The primary antibodies were listed as below: anti‐Cyclin D1 (ab16663; Abcam), anti‐MMP9 (ab76003; Abcam), anti‐Bax (ab32503; Abcam), anti‐NUCKS1 (ab80425; Abcam), and anti‐GAPDH (ab8245; Abcam). The membrane was mixed with the secondary antibody (Abcam) for 2 h and was detected by ECL system (Bio‐Rad).