Following 36 h of siRNA transfection, cells were then re-seeded in 10 cm dishes at ∼70% confluency and left to grow overnight in a 37°C incubator with 5% CO2. The following morning, safe and toxic ASOs were transfected at 80 nM into cells for 8 h using Lipofectamine 2000 (Life Technologies). Cells were washed with cold 1 × PBS and collected using a cell scraper. Cell lysate was prepared using RIPA buffer (ThermoFisher) and cleared by centrifugation at 10,000 g for 20 min at 4°C. Proteins (40 μg/lane) were separated by 4%–12% SDS-PAGE and transferred to membrane. Interested proteins were detected with specific antibodies, and visualized using ECL.
The following antibodies were purchased from Abcam: NAT10 (ab194297), DDX21 (ab182156), NPM1 (ab10530), NCL (ab134164), acetylated alpha-tubulin (ab24610), and cleaved PARP1 (ab4830). PSF (sc-374502) and P54 (sc-376865) antibodies were from Santa Cruz Biotechnology. The RNase H1 antibody (15606-I-AP) was obtained from ProteinTech. Quantification of proteins detected in Western blot was performed using ImageJ and normalized to the levels of GAPDH detected from the same blot using the GAPDH antibody (G9545; Sigma Aldrich). Anti-rabbit secondary antibody conjugated to HRP (170-6515) and anti-mouse secondary antibody conjugated to HRP (170-6516) were purchased from Bio-Rad.
The following antibodies were purchased from Abcam: NAT10 (ab194297), DDX21 (ab182156), NPM1 (ab10530), NCL (ab134164), acetylated alpha-tubulin (ab24610), and cleaved PARP1 (ab4830). PSF (sc-374502) and P54 (sc-376865) antibodies were from Santa Cruz Biotechnology. The RNase H1 antibody (15606-I-AP) was obtained from ProteinTech. Quantification of proteins detected in Western blot was performed using ImageJ and normalized to the levels of GAPDH detected from the same blot using the GAPDH antibody (G9545; Sigma Aldrich). Anti-rabbit secondary antibody conjugated to HRP (170-6515) and anti-mouse secondary antibody conjugated to HRP (170-6516) were purchased from Bio-Rad.