Molecular insights into DDX3X–mRNA regulation in castration-resistant prostate cancer
Molecular insights into DDX3X–mRNA regulation in castration-resistant prostate cancer
A total of 8 × 10⁶ cells were lysed in 1 mL of non-denaturing lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1.5 mM MgCl₂; and 0.25% NP-40) supplemented with 1× Halt protease and phosphatase inhibitor cocktail and divided for DDX3X pulldown assays. Protein concentrations were quantified to normalize input across cell lines, and each sample was adjusted to a final volume of 480 μL. An aliquot of 60 μL (1/8 of the total) from each sample was reserved for mRNA input normalization. Protein G Dynabeads, pre-bound to DDX3X or IgG control antibodies, were incubated with 480 μL of lysate for 4 h at 4 °C.  cells were cultured in RPMI 1640+L-glutamine medium, supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, and 0.2% Normocin, at 37℃, with 5% CO2.   RNA from all samples, including input controls, was extracted using the RNeasy Mini Kit, and cDNA was synthesized from the isolated RNA for input and DDX3X RIP samples as described above Libraries were prepared using the KAPA RNA HyperPrep Kit following manufacture's instructions 