Dylight 800 goat anti rabbit igg

Total proteins were extracted using RIPA (Beyotime, Shanghai, China) with 1 mM PMSF (Beyotime, Shanghai, China). Nuclear and cytoplasmic proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). Western blotting was conducted following previous procedures [21 (link)]. Antibodies used for western blotting and their dilutions were as follows: anti-DHX15 (1:1000, sc-271686, Santa Cruz Biotechnology, USA), anti-GFP (1:1000, sc-9996, Santa Cruz Biotechnology, USA), anti-NF-κB p65 (sc-8008, Santa Cruz Biotechnology, USA), anti-p-NF-κB p65 Ser 536 (1:1000, sc-136548, Santa Cruz Biotechnology, USA), anti-β-Actin (1:1000, sc-47778, Santa Cruz Biotechnology, USA), anti-cyclin D1 (1:1000, 2978s, CST, USA), anti-Lamin A/C (1:1000, 10298-1-AP, proteintech, USA), Dylight 800-goat anti-rabbit IgG (1:2000, A23920, Abbkine, USA), and Dylight 800-goat anti-mouse IgG (1:2000, A23910, Abbkine, USA).

Cell protein was extracted with RIPA lysis buffer supplemented with PMSF and phosphatase inhibitor cocktail A (P1801, Beyotime, Shanghai, China), and total protein was quantified using a BCA Protein Assay Kit (P0012S, Beyotime, Shanghai, China). Equal amounts of protein were loaded onto 10% SDS/PAGE gels and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Then, the membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), and the PVDF membranes were incubated with the primary antibody overnight at 4℃ followed by secondary antibody incubation. The primary antibodies used in this study include ENO1 (1: 5000; ab155102, Abcam, Cambridge, MA, USA); anti-E-cadherin (1:10,000; ab40772, Abcam, Cambridge, MA, USA), anti-N-cadherin (1:5000; ab76011, Abcam, Cambridge, MA, USA), anti-Vimentin (1:2000; ab92547, Abcam, Cambridge, MA, USA), and anti-β-actin (ab115777, 1:2000, Abcam, Cambridge, MA, USA). The secondary antibody Dylight 800 Goat Anti-Rabbit IgG (1:5000, A23920, Abbkine, Wuhan, China) was used for 1 h incubation at room temperature. Images were acquired by Odyssey CLX (LICOR, Lincoln, NE, USA), and band analyses were performed using IMAGE J software (NIH).

The cells were grown into six-well cell culture plates at 4 × 10⁵ cells/well and after 36 h of transfection, they were lysed with RIPA lysis and extraction buffer as described by the manufacturer (Thermo Fisher Scientific Inc, Rockford, IL, USA). The proteins were separated on 8% SDS-PAGE gels and electrotransferred to PVDF membrane (Millipore Co., Billerica, MA, USA). The PVDF membrane was blocked with 5% non-fat milk in TBS-T (0.15 mM sodium chloride, 0.01 mM Tris-base and 0.1% tween-20, pH 8.0) for 3 h, the PVDF membrane was incubated with rabbit anti-GFP polyclonal antibody (Abcam, Cambridge, UK) diluted in TBS-T(1:1000)overnight at 4 ℃. After washing three times with TBS-T, the PVDF membrane was incubated with DyLight 800 goat anti-rabbit IgG (Abbkine, Wuhan, China) at 1:8000 dilution in TBS-T. Finally, the membrane was washed three times with TBS-T, and bands were scanned using the Odyssey system (LI-COR Bioscience, Lincoln, NE, USA).