Anti flag magnetic beads

The in vitro kinase assay was performed as described previously^{51 (link)}. Briefly, Flag-TGF-β1WT or Flag-TGF-β1T282A was transiently overexpressed with Myc-PRKACA in HEK293T cells and purified using anti-Flag magnetic beads or anti-Myc magnetic beads (88842, Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. The precipitated Flag-TGF-β1, Flag-TGF-β1T282A, and Myc-PRKACA proteins were resuspended in 40 μl of 1 × kinase buffer (9802) supplemented with 200 μM ATP (9804) (Cell Signaling). The reaction was carried out for 30 min at 30 °C and was terminated by the addition of 20 μl 3 × SDS sample buffer. Each sample was then boiled for 10 min at 100 °C and subjected to SDS–PAGE and IB analysis using the indicated antibodies.

SW480 cells were cultured in 60-mm culture dishes and transfected with Ets1 and Sp1 plasmids using Lipofectamine 3000 reagent (Invitrogen). After transfection for 24 h, the cells were processed for coimmunoprecipitation by standard procedures as previously described in western blot analysis. The cell extract was used to immunoprecipitate Flag using anti-Flag magnetic beads (A36798; Thermo Fisher) according to the manufacturer’s instructions, and the immunoprecipitates were analyzed by western blot analysis using anti-HA, anti-GST and anti-Flag antibodies.

Cells were resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, and 1 mM EDTA) with protease and phosphatase inhibitor cocktails (cat. no. 04693132001 and 04906837001, respectively; Roche, Basel, Switzerland). After incubation for 30 min on ice, the cells were centrifuged at 13,000 g for 15 min at 4 °C to remove cellular debris. Proteins in the whole-cell lysate were quantified with a bicinchoninic acid assay kit (cat. no. 23225; Thermo Fisher Scientific).
For immunoprecipitation (IP) of Flag- or Myc-tagged proteins, cleared lysate was mixed with anti-Flag magnetic beads (cat. no. M8823; Sigma-Aldrich) or anti-Myc magnetic beads (cat. no. B26301; Bimake, Houston, TX, USA) for 2–4 h at 4 °C. For IP of endogenous NONO or PRMT1 protein, cleared lysate was incubated overnight at 4 °C with 0.5–2 μg of anti-NONO antibody (cat. no. 611279; BD Bioscience), anti-PRMT1 antibody (cat. no. 2449; CST), or isotype-matched IgG. Dynabeads Protein G (Invitrogen, Carlsbad, CA, USA) were then added, and the tube was rotated for 2–4 h at 4 °C. The beads were washed three times with RIPA buffer and incubated with 0.1 M glycine HCl (pH 3.1) to release immunoprecipitated proteins. The supernatant was neutralized with 1 M NaOH and analyzed by western blotting.