Nucleolin

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).

Cells were lysed in SDS-PAGE protein loading buffer 1× (60 mM TrisHCl (pH 6.8), 10% glycerol, 2% SDS and 0.1% bromophenol blue) or assay buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and protease inhibitors). Proteins were analyzed by SDS-PAGE and transferred onto PVDF membranes, which were blocked with 3% BSA in PBS-buffered saline (PBS) containing 0.1% Tween 20 and incubated overnight at 4 °C with primary antibodies. After incubation with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature, membranes were revealed with the ECL system (Lumi-Light Western Blotting Substrate, Roche). Images were acquired by ImageQuant™ LAS 4000 mini Image Analyzer (Fujifilm, Tokyo, Japan) and quantified using ImageJ software. TUBULIN loading control was used when required. The primary antibodies used were the following: USP48 (Abcam, Cambridge, UK; ab72226, 1:1000), p65 (Cell Signaling Technology, Danvers, MA, USA; 8242, 1:1000), IκBα (Cell Signaling Technology, Danvers, MA, USA; 4814, 1:1000), P-IκBα (Cell Signaling Technology, Danvers, MA, USA; 2859, 1:1000), Cyclin E (Abcam, Cambridge, UK; ab2094; 1:1000), PCNA (Abcam, Cambridge, UK; ab29; 1:1000), NUCLEOLIN (Abcam, Cambridge, UK; ab70493; 1:1000); TUBULIN (Sigma-Aldrich, St. Louis, MO, USA; T5168, 1:1000).

Protein samples consisting of five fish/sample were collected at appropriate stages. Embryos were homogenized and suspended in sample buffer containing Tris-HCl pH 8.0, NaCl, SDS, sodium deoxycholate, NP-40 and protease inhibitor and used for western blotting according to standard protocols (Dash et al., 2021 (link)). Protein quantity was estimated via a bicinchoninic acid (BCA) assay. The primary antibodies used were: γ-tubulin (1:1000, Sigma-Aldrich, T6557), α-tubulin (1:10,000, Sigma-Aldrich, T5168), p53 (1:500, Cell Signaling Technology, 2524S) and Nucleolin (1:500, Abcam, ab22758). Western blots were imaged and quantified using a CLx-Scanner (Li-COR) and Odyssey Software. For quantification, band intensities for p53 were compared with the housekeeping control protein γ-tubulin. Two-tailed, paired Student's t-test was performed for statistical analysis.

Cells were seeded on coverslips in a 24-well plate, washed with PBS (Biological Industries, Israel), and fixed with 4% formaldehyde in PBS for 25 min at room temperature. After fixation, cells were washed with PBS, and permeabilized and blocked in PBS containing 0.2% Triton X-100% and 1% BSA. Slides were incubated with primary antibodies to UBF (Sigma/Santa Cruz, CA, USA), Fibrillarin (Abcam, Cambridge, UK), NPM (Abcam, Cambridge, UK), Nucleolin (Abcam, Cambridge, UK), RPA194 (Santa Cruz, CA, USA), PICT-1 (Santa Cruz, CA, USA), PML (Santa Cruz, CA, USA), ORF45 (Santa Cruz, CA, USA), ORF65 [64 (link)] (a kind gift from Shou Jiang Gao) or ORF59 (a kind gift from Prof. Bala Chandran, University of South Florida) [65 (link)] at 40 C, followed by incubation with secondary conjugated antibodies (Rhodamine, Cy3, Alexa Fluor 647 or Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h at room temperature. To stain the nuclei, cells were incubated for 10-min with 0.05 µg/mL Hoechst dye (Sigma) in PBS. The slides were mounted with anti-fading medium (1% n-Propyl gallate, 90% Glycerol in PBS). Cells were examined and photographed under a confocal laser-scanning microscope (Leica SP8 Confocal Live Microscope).