Anti srsf1

Proteins in cells were extracted using RIPA lysis buffer (Beyotime), and concentrations were determined using a protein assay kit (Beyotime). Protein bands were scanned using the Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA). The following antibodies were used: anti-PCNA, anti-PTK2 (Abclonal), anti-SRSF1, anti-MMP2, and anti-MMP9 (Santa Cruz Biotechnology). RIP assays were performed using the BersinBio^TM RNA Immunoprecipitation (RIP) Kit (BersinBio), with anti-SRSF1 (Santa Cruz Biotechnology), anti-FLAG (Abclonal), and appropriate control IgG (BersinBio) antibodies. Subsequently, qRT-PCR and agarose gel electrophoresis assays were performed. IHC was performed using antibodies against PCNA, Ki-67, PTK2, and MMP2 (Abclonal). IF was performed using an antibody against SRSF1 (Santa Cruz Biotechnology). Images were taken using a microscope (Leica Microsystems). All assays were performed according to the manufacturer’s protocol. The catalog numbers for all antibodies used were provided in Table S2.

RNA pulldown analysis was performed as previously described [30 (link)]. In brief, streptavidin agarose conjugate (Millipore, Burlington, MA, USA, 16-126) was used to covalently link biotin labeled RNA, followed by incubation with cellular extract at 4 °C for 4 h in buffer D (20 mM Tris–Cl, 300 mM KCl, 2 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF, pH 7.5). Immunoblotting analysis was performed using anti-SRSF9 (Invitrogen, Carlsbad, CA, USA, MA5-26990), anti-SRSF1 (Santa Cruz Biotechnology, Dallas, TX, USA, SC-33652) and anti-SRSF3 antibody (Invitrogen, 334200) as previously described [30 (link)].

The nuclear fractions of proteins for western blotting were prepared from cell cultures using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Proteins were separated on a 10 % SDS–polyacrylamide gel and transferred onto the membrane. The membrane was incubated with appropriate primary antibody: anti-SRSF1 (Santa Cruz), anti-SRSF2 (Abcam), or anti-β-actin (Abcam), and washed and incubated with secondary antibody (Dako). The membrane was developed using TMB reagent (Sigma-Aldrich). All data were quantitated using MultiGauge software (Fujifilm). β-Actin expression was used for data normalization.