2.2. Cell transfection The siRNA duplexes targeting human RNF181 (si-RNF181) and the scrambled control (si-NC), together with the overexpression plasmids encoding full-length human RNF181 (oe-RNF181) and the corresponding empty-vector control (oe-NC), as well as the ubiquitin constructs HA-Ub, HA-K48-Ub, HA-K63-Ub, and the autophagy-related plasmid ATG7-Flag, were all synthesized by Beijing Tsingke Biotech Co., Ltd. (Beijing, China). Transfection of A549, PC-9, and 293 T cells (70–80 % confluence) was carried out with 50 nM siRNA or 2 μg plasmid DNA per well (6-well plate format) using UltraFection 3.0 transfection reagent (4A Biotech, Suzhou, China) in antibiotic-free medium; assays were performed 24–48 h post-transfection.
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