Ab87433

Protein concentration was measured using the Protein Assay Dye Reagent Concentrate (Bio-Rad). Protein lysates were separated on 10% SDS-PAGE gels, transferred onto nitrocellulose membrane (Amersham) and blocked for 1 h in 5% BSA–TBS. After incubation with primary antibodies (overnight) and secondary antibodies (1h, RT), the membranes were washed and analyzed using the LI-COR Odyssey Infra-red Imaging System. Immunoreactivity bands were with Image J software or by the software included in the Odyssey Infra-red Imaging System. Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti-FLAG M2 antibody (Sigma Aldrich, F1804, 1:2000), PRPF8 (Abcam, ab87433; 1:300), EFTUD2 (Abcam, ab72456, 1:200), ZNHIT2 (Abcam, ab126133; 1:500), RUVBL1 (Cell signaling #12300; 1:500), RUVBL2 (Cell signaling #8959; 1:500), PIH1D1 (Invitrogen #PA5-61482, 1:1000), RPAP3 (Invitrogen #PA5-58334D; 1:500). Secondary antibodies used were as follows: Anti-Rabbit IgG (H + L) (DyLightTM 680 Conjugate) (Cell signaling #5366; 1:15000) and Anti-Mouse IgG (H + L) (DyLightTM 800 4X PEG Conjugate) (Cell signaling #5257; 1:15000). Conjugated antibodies used were StrepMAB-Classic HPR (IBA Lifesciences #2-1509-001; 1:30000) and Anti-polyHistidine-Peroxidase (Sigma Aldrich #A7058; 1:1000).