Sf3b1

Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, blocked, and then incubated at 4 °C overnight with the following antibodies: SR140 (at the dilution of 1:150, Santa Cruz, sc-398718); SF3A2 (at the dilution of 1:150, Santa Cruz, sc-390444); SF3B1 (at the dilution of 1:150, Santa Cruz, sc-514655); SYF1 (at the dilution of 1:150, Santa Cruz, sc-271037); or STRAP (at the dilution of 1:200, Bethyl, A304-735A). After washing, cells were incubated with goat anti-rabbit Alexa Fluor 488 antibody (at the dilution of 1:150, Life Technologies, A-11008) or goat anti-mouse Alexa Fluor 555 antibody (at the dilution of 1:150, Life Technologies, A-21422) and counter-stained with DAPI to detect nuclei.

Proteins were separated by 10% SDS-PAGE and probed with primary antibodies. Primary antibodies, at the dilution of 1:1000 for each, included: SNRPA (Santa Cruz, sc-376027); U2A’ (Santa Cruz, sc-393804); SR140 (Santa Cruz, sc-398718); HELIC2 (Santa Cruz, sc-393170); PRPF3 (Santa Cruz, sc-101130); SYF1 (Santa Cruz, sc-271037); SF3A3 (Santa Cruz, sc-393673); SF3A2 (Santa Cruz, sc-390444); SF3B1 (Santa Cruz, sc-514655); STRAP (BD Transduction Labs, #611346); DDX15 (Santa Cruz, sc-271686); TFIIIC110 (Santa Cruz, sc-81406); GAPDH (Cell Signaling, #2118), and CHERP (Santa Cruz, sc-100650). β-Actin (Sigma, A5316) was used at the dilution of 1:10,000.

The protocol was adapted from the RIP–ChIP protocol described previously^{45 (link)}. Briefly, protein A Sepharose beads (Sigma) were coated with 3 µg of U1 snRNP, SF3B1, SRSF1, U2AF2 or mouse IgG antibody (Santa Cruz), followed by incubation with 2 mg of Hep3B or SNU398 total cell lysates overnight. The RNA–protein–bead complexes were washed once with NT2 crowders (25 mg Ficoll PM400 (GE Healthcare), 75 mg Ficoll PM70 (GE Healthcare) and 2.5 mg dextran sulfate (Fluka) in 10 ml of NT2 buffer) and five times with NT2 buffer (50 mM Tris pH 7.0, 150 mM NaCl, 1 mM MgCl₂ and 0.05% (v/v) NP-40). Protein–RNA complexes were collected in 100 μl of NET2 buffer (1 mM DTT, 16.7 mM EDTA, 200 U RNaseOUT (Thermo Fisher) and 100 U SUPERase In (Ambion) in 1× NT2 crowder), supplemented with 100 μl of 2× SDS–TE (100 mM Tris pH 7.5, 10 mM EDTA pH 8.0 and 1% SDS). RNA was isolated using TRIzol reagent and subsequently purified with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1).