Ab183660

Total protein was extracted by RIPA buffer (Beyotime, Shanghai, China) involved in protease inhibitors. The protein concentration measured by BCA protein assay kit (Thermo Fisher Scientific). The supernatant soluble lysate was mixed with loading buffer and the solution boiled for 10 min and stored at −20°C. Isolated proteins were electrophoresed on 8% SDS-PAGE electrophoresisand transferred to a 0.22-μm PVDF membrane and blocked with 5% nonfat milk. Afterward, the membranes were then incubated with primary antibodies against GAPDH (1:5000, ab181602, Abcam, Shanghai, China), ABHD2 (1:1000, ab230417, Abcam, Shanghai, China), NUDT21 (1:3000, ab183660, Abcam, Shanghai, China), Caspase-3 (1:1000, ab13847, Abcam, Shanghai, China) and Cleaved Caspase-3 (1:1000, ab49822, Abcam, Shanghai, China) overnight at 4°C. The membranes were then incubated with the HRP-conjugated secondary antibody (1:10,000, Santa Cruz) and the protein level detected by chemiluminescence.

10 µg of total protein lysates were loaded into 10% 37.5:1 bis-acrylamide SDS-PAGE gels. SDS-PAGE gels were transferred onto PDVF membranes and targeted proteins were visualized with a chemiluminescence system and subsequent imaging with an X-ray developer. Antibodies used to detect protein expression levels are as follows: CSTF2 (abcam: ab72297), CPSF3 (abcam: ab72299), CPSF6 (Santa Cruz: sc-100692), CPSF7 (Bethyl Laboratories: A301-360A), NUDT21 (abcam: ab183660), hnRNP L (abcam: ab6106), hnRNP A3 (abcam: ab78300), RBM3 (Proteintech: 14363-1-AP).

Cells were lysed using RIPA buffer (P0013B, Beyotime Biotechnology). Subsequently, total protein was separated using SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked and were subsequently incubated at 4 °C overnight with the following primary antibodies against: GAPDH (2118, 1:1000; CST.), beta-Tubulin (16,305, 1:2000; CST), NDUFS2 (sc-390596, 1:1000; MDL Biological, Inc.), AKT (ab8805, 1:1000; Abcam), P-AKT308(ab38449, 1:1000; Abcam), NUDT21 (ab183660, 1/1000; Abcam), PI3K(#4257, 1/1000; CST), and P-PI3K(#4228, 1/1000; CST). All antibodies were used according to the manufacturer’s protocol. Following primary incubation, membranes were incubated with secondary antibodies, HRP-conjugated goat anti-rabbit IgG (PR30011, 1:20,000; Proteintech Co., Ltd.) and HRP-conjugated goat anti-mouse IgG (PR30012, 1:20,000; Proteintech Co., Ltd.) at room temperature for 1 h. Proteins were visualized using ECL Reagent (42,029,053, Millipore Sigma).