Anti rna polymerase 2 ctd repeat ysptsps phosphos2

Cells were lysed in urea lysis buffer (50 mM Tris-HCl pH7.9, 10 mg/mL CHAPS, 480 mg urea) supplemented with protease inhibitor cocktail (Aprotinin/Leupeptin/Pepstatin (A/L/P), PMSF, and sodium butyrate), pre-cleared by centrifugation and subjected to standard Western blotting procedures. The following primary and secondary antibodies were used: anti-TOP1 (Bethyl, Cat #: A302-590A, 1:1000 or 1:2000), anti-RNA polymerase II CTD repeat YSPTSPS (PhosphoS2) (Abcam, Cat #: ab5095, 1:1000), anti-HA (HA.11) (Biolegend, Cat #: 901501, 1:4000), anti-actin (C-4) (Santa-Cruz, sc-47778, 1:1000 or 1:2000), anti-AFF4 (Proteintech 14662-1-AP, 1:20,000), anti-gamma H2A.X (phospho S139) antibody (Abcam, ab2893, 1 μg/mL), goat anti-Rabbit IgG HRP-Conjugated (Bethyl, Cat #: A120-201P, 1:20,000), goat anti-Mouse IgG HRP-Conjugated (Bethyl, Cat #: A90-516P, 1:20,000). The antibodies are also summarized in Supplementary Data 3.

For immunofluorescence staining of nuclear proteins, cells were fixed, permeabilized, and blocked with BSA as described above. The cells were incubated with the following primary antibodies in 1% BSA and 0.03% Triton X-100 in PBS (–) at 4 °C overnight: anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (5095, Abcam; 1:500), anti-topoisomerase I (85038, Abcam; 1:500), anti-RPA194 monoclonal (48385, Santa Cruz Biotechnology; 1:100), anti-fibrillarin monoclonal (2639, Cell Signaling Technology; 1:500), and anti-NPM 1 monoclonal (32–5200, Invitrogen; 1:500) antibodies. After washing with PBS (–), the cells were incubated with the following secondary antibodies in 1% BSA and 0.03% Triton X-100 in PBS (–) at room temperature for 1 h: Goat Anti-Rabbit IgG H&L-Alexa Fluor^® 568 (175471, Abcam; 1:500) and Goat Anti-Mouse IgG H&L-Alexa Fluor^® 568 (175473, Abcam; 1:500). For nuclear staining, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) at 1:500 at room temperature for 1 h. For RNA staining, cells were incubated with StrandBrite^TM (AAT Bioquest) at 1:4000 at room temperature for 30 min. For cell viability assays, cells were incubated with 1 μM SYTOX Orange^TM (Thermo Fisher Scientific) and 20 μg/mL bisBenzimide H 33342 trihydrochloride (Hoechst33342; Sigma-Aldrich) in complete medium at 37 °C for 30 min prior to the observation.