Trueblot anti rabbit igg hrp

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).

Immunoprecipitation was performed as described previously.^{26 (link)} In brief, cell lysates were mixed with anti-IRF9 antibodies and protein A magnetic FG beads (Tamagawa Seiki, Nagano, Japan) prewashed with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, 0.25% gelatin, and 0.02% sodium azide), and incubated with rotation overnight at 4 °C. The beads were then separated with a magnetic separator and mixed with 2× SDS sample buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, and 0.02% Bromophenol Blue) and boiled using a heat block at 95 °C for 3 min to elute the proteins. For western blotting, 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.

Western blotting was performed as previously described69 (link). Briefly, lysate samples were separated on 5–20% gradient polyacrylamide gel by SDS–PAGE following electrical transfer to PVDF membranes (GE Healthcare). 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. HRP-conjugated corresponding secondary antibodies (GE Healthcare) were subsequently used. Trueblot anti-rabbit IgG HRP (Rockland, Limerick, PA, USA, 1:1,000) was used as the secondary antibody to avoid interfering immunoprecipitated immunoglobulin heavy and light chains. Bound antibodies were detected using Immunostar LD reagents (Wako). The following antibodies were used: Hnrnp-U (#ab180952, 1:1,000) and Nucleolin (#ab22758, 1:1,000) from Abcam (Cambridge, UK); HA tag (#561, 1:10,000) and Syncrip (#RN046PW, 1:1,000) from MBL; Hnrnp-A2/B1 (#R4653, 1:1,000) and β-actin (#A1978, 1:2,000) from Sigma-Aldrich; and YBX1 (D299, 1:1,000), Igf2BP1 (D33A2, 1:1,000) and HnrnpA1 (D21H11, 1:1,000) from Cell Signaling Technology (CST, Danvers, MA, USA).