Anti tata binding protein

The cell fractionation protocol was adapted from the Ficoll-digitonin protocol previously described (44 (link)). Briefly, 1.5 × 10⁶ trypsinized WT A549 cells were washed with PBS twice and resuspended in 600 μl of HEPES-sucrose-Ficoll (HSF) solution (20 mM HEPES-KOH, 6.25% Ficoll, 0.27 M sucrose, 3 mM CaCl₂, 2 mM MgCl₂) with 50-μg/ml digitonin and EDTA-free protease inhibitor cocktail (Roche). The cells were kept on ice for 10 min, before they were spun down at 1,000 × g for 3 min. The supernatant was collected and then further centrifuged at 15,000 × g for 10 min to generate the cytosol fraction. The nucleus pellet from the first centrifugation was rinsed with HSF buffer once and spun down again at 1,000 × g for 3 min. The supernatant was collected as the washed fraction, and the pellet was then lysed in 50 μl of RIPA buffer with EDTA-free protease inhibitor cocktail (Roche) on ice for 20 min. The lysed nuclei were then centrifuged at 10,000 × g for 10 min, before the supernatant was collected as the nuclear fraction for Western blot analysis. The antibodies used for this experiment included mouse anti-alpha-tubulin antibody (clone DM1A; catalog number 14-4502-82; Thermo Fisher), anti-TATA-binding protein (catalog number 8515s; Cell Signaling Technology), and mouse anti-RTFDC1 antibody (clone 1E8; catalog number LS-C340588; LifeSpan Biosciences).