Total protein extraction kit prottot 1kt

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.

Proteins were firstly isolated with protein lysis buffer (Keygen, Nanjing, China) and extracted by Total Protein Extraction Kit (PROTTOT-1KT, Sigma-Aldrich, USA). Later, the proteins extracted were treated with SDS Quick Match Gel Kit (P0670-250 ml, Beyotime Biotechnology, Shanghai, China) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Vicmed, China). Afterwards, the membranes blocked by 5% skimmed milk were co-cultivated with the primary antibodies, including Anti-β-actin, Anti-CLDN12and Anti-SRSF1 antibodies, at 4°C overnight. Subsequently, secondary antibodies were added for incubation. Eventually, the expressions of target proteins were analysed by J software. β-actin served as the internal reference.