Snrnp200

Western blotting was performed as previously described [29 (link)]. To confirm the proteins identified by LC-MS/MS, RNA immunoprecipitation samples were transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Chalfont St. Giles, UK) after SDS-PAGE. In other cases, lysate samples were separated by 10–20% gradient SDS-PAGE followed by electrical transfer to PVDF membranes. After blocking with 5% dry milk, membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo Bio, Tokyo, Japan) overnight at 4° C. Horseradish peroxidase (HRP)-conjugated corresponding secondary antibodies (GE Healthcare) were subsequently used. Trueblot anti-rabbit IgG HRP (1:1000; Rockland, Limerick, PA, USA) was used as the secondary antibody to avoid interfering with the immunoprecipitated immunoglobulin heavy and light chains. Bound antibodies were detected using Immunostar LD reagents (Wako). The following antibodies were used: HBV PreS2 (#ab30923, 1:1000), DNA-PK (#ab32566, 1:1000), DHX9 (#ab26271, 1:1000), ILF3 (#ab92355, 1:1000), and snRNP200 (#ab118713, 1:1000) from Abcam (Cambridge, UK); leucine-rich PPR motif-containing protein (LRPPRC; #ARP41093, 1:1000) from AVIVA Systems Biology (San Diego, CA, USA); and β-actin (#5125, 1:2000) from Cell Signaling Technology (Danvers, MA, USA).

In the presence of RIPA lysis buffer, cells are lysed and total proteins are extracted. Basic experiment procedures of Western blot were consistent with our previous methods [11 (link)]. The introductions of primary antibodies were as follows: SNRNP200 (Cat No. ab176715, Abcam), CyclinA (Cat No. SAB4503499, Sigma-Aldrich), CDK2 (Cat No. 05–363, Merck Millipore), CyclinD (Cat No. 05–137, Merck Millipore), CDK1 (Cat No. 19532-I-AP, Protech), p21^WAF1/Cip1 (cyclin-dependent kinase inhibitor 1A), (Cat No. 10355-I-AP, Protech), p53 (Cat No.10442-I-AP, Protech), CyclinB (Cat No.55004-I-AP, Protech) and CyclinE (Cat No. 05–363, Merck Millipore, Darmstadt, Germany). The housekeeping protein we used was Histone H3 (Cat No. Sc-10,809, SantaCruz) or beta-actin (β-actin; Cat. No. ab129348, Abcam, Cambridge, UK). Image J was applied to the quantitative analysis of proteins.