Sc 3879

As described previously [70 (link)], RIP was performed using 16.5 dpc mouse ovaries. The ovaries were lysed in cell lysis buffer (P0013, Beyotime, Shanghai, China) containing PMSF (1:100, ST506, Beyotime, Shanghai, China), a proteinase inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China), DTT (1:50, ST041-2 ml, Beyotime, Shanghai, China), and an RNase inhibitor (1:20, R0102-10 kU, Beyotime, Shanghai, China). After incubation on ice for 20 min, the lysate was added to 20 μl of protein A agarose (P2051-2 ml, Beyotime, Shanghai, China) for preclearing at 4 °C for 1 h. Two micrograms of an SRSF1 antibody (sc-33652, Santa Cruz Biotechnology, California, USA) and a normal mouse IgG (sc-3879, Santa Cruz Biotechnology, California, USA) were added to the lysate, followed by overnight incubation at 4 °C. The next day, 60 μl of protein A agarose was added to the lysate followed by incubation at 4 °C for 4 h. The agarose complexes containing antibodies, target proteins, and RNA were washed for 5 min at 4 °C, which was repeated 3 times. Protein-bound RNA was then extracted using RNAiso Plus and the Direct-zol RNA MicroPrep kit. RIP–qPCR was performed according to the above RT–qPCR protocol.

Total protein was extracted with cell lysis buffer (P0013, Beyotime) containing PMSF (1:100, ST506, Beyotime) and a protease inhibitor cocktail (1:100, P1005, Beyotime). After incubation on ice for 20 min, the lysate was added to 20 μl of protein A agarose (P2051-2 ml, Beyotime) for pre-clearing at 4°C for 1 hr. Two micrograms of an SRSF1 antibody (sc-33652, Santa Cruz Biotechnology) and a normal mouse IgG (sc-3879, Santa Cruz Biotechnology) were added to the lysate and the mixture was incubated overnight at 4°C. The next day, 60 μl of protein A agarose was added to the lysate, which was then incubated at 4°C for 4 hr. The agarose complexes containing antibodies and target proteins were washed three times for 5 min at 4°C. IP and Co-IP were performed according to the above western blotting protocol. The complex was sent to the protein mass spectrometry laboratory for IP-MS analyses using a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Scientific, Waltham, MA, USA). Raw data from the mass spectrometer were preprocessed with Mascot Distiller 2.4 for peak picking. The resulting peak lists were searched against the uniport mouse database using Mascot 2.5 search engine.

As described previously (Gagliardi and Matarazzo, 2016 (link)), RIP was performed using 5 dpp mouse testes. The testes were lysed in cell lysis buffer (P0013, Beyotime) containing PMSF (1:100, ST506, Beyotime), a proteinase inhibitor cocktail (1:100, P1005, Beyotime), DTT (1:50, ST041-2 ml, Beyotime), and an RNase inhibitor (1:20, R0102-10 kU, Beyotime). After incubation on ice for 20 min, the lysate was added to 20 μl of protein A agarose (P2051-2 ml, Beyotime) for pre-clearing at 4°C for 1 hr. Two micrograms of an SRSF1 antibody (sc-33652, Santa Cruz Biotechnology) and a normal mouse IgG (sc-3879, Santa Cruz Biotechnology) were added to the lysate, which was then incubated overnight at 4°C. The next day, 60 μl of protein A agarose was added to the lysate, and the mixture was incubated at 4°C for 4 hr. The agarose complexes containing antibodies, target proteins, and RNA were washed three times for 5 min at 4°C and repeated. Protein-bound RNA was extracted using RNAiso Plus and a Direct-zol RNA MicroPrep Kit. RIP-qPCR was performed according to the above RT-qPCR protocol.