Immunostaining of Splicing Factors in Human Cell Lines

Cells were grown on 18 mm coverslips, washed with phosphate-buffered saline (PBS) and fixed for 20 min in 4% PFA. U2OS Tet-On cells were then permeabilized in 0.5% Triton X-100 for 2 min, after which they were blocked in bovine serum albumin (BSA). HFF-1 and HepG2 were permeabilized twice in 0.1% Triton X-100 for 5 min. Cells were immunostained for 1 hr with a primary antibody, and after subsequent washes the cells were incubated for 45 min with secondary fluorescent antibodies. Primary antibodies: Mouse anti-Serine and Arginine Rich Splicing Factor 2 (SRSF2) (which marks SRRM2 [52 (link)]), rabbit anti-SON (Sigma), rabbit anti-Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) (Abcam), rabbit anti-SRRM2 (Abcam) and rabbit anti-Serine and Arginine Rich Splicing Factor 7 (SRSF7) (Santa Cruz). Secondary antibodies: Alexa647-labeled goat anti-mouse IgG, Alexa594-labeled goat anti-mouse IgG, Alexa647-labeled donkey anti-rabbit IgG (Life Technologies, Thermo Fisher Scientific), dyLight488-labeled goat anti-rabbit IgG and Alexa488-labeled goat anti-mouse IgG (Abcam). The cells were mounted in mounting medium.