Ab204916

IHC was performed with anti-SC35 antibody (abcam, ab204916) as previously described (44 (link)). Western blot was carried out as previously described (45 (link)). The indicated primary antibodies used in this study were listed in Table S6. In vivo CLIP of SLMAP exon 25 or CETN3 exon 6 pre-mRNA bound to SRSF1, SRSF2, and hnRNPM proteins were performed as described previously (2 (link)). Briefly, hemagglutinin-tagged SRSF1, hnRNPM, SRSF2, and its domain deletion mutants or empty vector control were transfected into 293T cells transiently, and ultraviolet cross-linking was performed followed by immunoprecipitation using Magna RIP kit (Millipore).

The cells were cleaned with 1 × PBS before being lysed with M-PER mammalian protein extraction reagent (78501, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with a Cocktail (11873580001, Roche, Basle, Switzerland). Protein concentrations were measured as indicated by the manufacturer using the BCA protein assay kit (23250, Thermo Fisher Scientific, Waltham, MA, USA).
Protein samples (30 μg) were separated in SDS-PAGE gels ranging from 4% to 15% and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, San Francisco, CA, USA). After blocking in 5% skim milk for 60 min, the membranes were treated with the appropriate primary antibodies and fluorescent-conjugated secondary antibodies. Primary antibodies against SRSF2 (ab204916, 1:1000, Abcam, Cambridge, UK), SLC7A11 (A2413, 1:1000, ABclonal, Wuhan, China), ACSL4 (A6826, 1:1000, ABclonal, Wuhan, China), and GPX4 (A1933, 1:1000, ABclonal, Wuhan, China) were used. The internal standard was probed with an anti-β-actin-peroxidase monoclonal antibody (A3854, Sigma-Aldrich, Bedford, MA, USA). In a Tanon 5200 MultiImage System (Tanon Science & Technology, Shanghai, China), ECL reagents (34080, Millipore, Burlington, MA, USA) were used to observe the bands. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to calculate band intensities.

Mouse monoclonal antibodies against firefly luciferase (sc-74548) and U2AF65 (sc-53942) were from Santa Cruz Biotechnology (Heidelberg, Germany). A mouse monoclonal antibody against GAPDH and rabbit monoclonal antibodies against SF3B1 (ab170854) and SRSF2 (SC35, ab204916) were purchased from Abcam (Cambridge, UK). A mouse monoclonal antibody against Streptag II was from Novagen^® (Sigma-Aldrich, Taufkirchen, Germany).